Interspecific micronuclear transplantation in Paramecium ...Interspecific micronuclear transplantation in Paramecium: nucleogenesis and stomatogenesis in asexual and sexual reproduction

Development 103, 179-191 (1988)Printed in Great Britain © The Company of Biologists Limited 1988

179

Interspecific micronuclear transplantation in Paramecium:

nucleogenesis and stomatogenesis in asexual and sexual reproduction

M. F. CHAU and STEPHEN F. NG

Zoology Department, University of Hong Kong, Hong Kong

Summary

The micronucleus of Paramecium plays an essentialrole in the development of the oral apparatus in bothasexual and sexual cycles. The present study analysesthis somatic function of the micronucleus by inter-specific transplantation of the micronucleus betweentwo species, P. jenningsi and P. tetraurelia. The twospecies are similar in nucleogenesis in the sexual cycle,in the dependence of stomatogenesis on the micronu-cleus and in the pattern of the oral ciliature. P.jenningsi, however, has a longer oral apparatus.Renucleated cell lines were derived from hetero-specific transplantation (P. jenningsi amicronuclcatesimplanted with micronuclei of P. tetraurelia), and alsofrom homospecific transplantation (P. jenningsi).Both homo- and heterospecific transplants exhibitedabnormal micronuclear propagation during cell div-ision. In the sexual cycle, the heterospecific trans-plants exhibited more severe micronuclear anomalies,suggesting interspecific incompatibility. On the otherhand, the stomatogenic consequences of the two types

of transplants in the asexual and sexual cycles weresimilar. It is concluded that micronuclear functions, inthe assembly and normal patterning of the oralciliature in the sexual cycle, are not species-specific.However, the oral apparatuses developed by thehomo- and heterospecific transplants were similar inlength, and approaching that of normal P. jenningsi.Hence, even though the micronucleus is necessary fordeveloping normal oral length, the oral length charac-teristic of a species is determined by species-specificnonmicronuclear factors. The present findings re-semble heterospecific dermal-epidermal inductive in-teractions in multicellular development, with themicronucleus exerting a nonspecies-specific 'intra-cellular inductive stimulus' on the oral anarchic fieldto promote oral development.

Keywords: Paramecium, micronucleus, stomatogenesis,interspecific transplantation, induction.

Introduction

The micronucleus and the macronucleus of ciliatedprotozoa are traditionally thought to be demarcatedsharply in terms of their functions. The micronucleusis obviously indispensable in nuclear reorganizationduring sexual reproduction, while the macronucleusgoverns somatic functions and asexual propagation.This traditional view has been critically reexamined,and the sharp functional demarcation between themicronucleus and the macronucleus is no longertenable in view of compelling evidence demonstratingthe somatic function of the micronucleus (reviewedby Ng, 1986). In particular, in Paramecium tetra-urelia, it has been shown that the micronucleus playsan important role in the development of the oralapparatus, in both asexual and sexual reproduction

(Ng & Mikami, 1981; Ng & Newman, 1984ft; Tarn &Ng, 1986; Chau & Ng, 1988a,ft,c). These studies havemade use of (1) amicronucleate cell lines generatedby removal of the micronucleus with a microinjectionneedle, (2) defective-micronucleus cell lines gener-ated by creating lesions in the micronucleus with lasermicrobeam irradiation of the micronucleus, or treat-ment of the cells with ds-dichlorodiammineplatinumII and also (3) haploids.

An amicronucleate cell line can be obtained follow-ing the removal of the two micronuclei from the cell.Such cell lines enter a growth depression periodmanifested by the development of defective, butmostly functional oral apparatuses, characterized byabnormal oral ciliature ('membranelles') and re-duction of oral length. These cell lines can graduallyrecover and the oral apparatus returns to a near-

180 M. F. Chau and S. F. Ng

normal condition. On the other hand, amicronucleatecells are doomed when induced to go through thesexual cycle (autogamy or conjugation), during whichthey resorb the preexisting oral apparatus, as usual,but fail completely to develop a new one. Stomato-genesis in the sexual cycle is arrested characteristi-cally at an early stage of alignment of the basal bodiesof the oral anarchic field into parallel rows, inpreparation for the formation of the oral membra-nelles ('initiation'); no oral structures can sub-sequently develop, resulting in astomy and death. Ofparticular interest, this specific stomatogenic functionrelies on the activity of micronuclear postmeioticdivisional derivatives, some of which reside in a'paroral cone area' that is to the immediate right ofthe oral anarchic field, at the time when oral membra-nelle assembly is initiated (see Ng & Newman,1984a). Cells possessing defective micronuclei areable to initiate the assembly of oral membranelles inthe sexual cycle, but the oral apparatus subsequentlydeveloped is frequently defective. We have con-cluded from these observations that the micronucleusplays an important, but replaceable, role in thedevelopment of a normal oral membranellar patternand length during asexual propagation, but its func-tion in the initiation of oral membranelle assembly inthe sexual cycle is indispensable. The studies ofdefective-micronucleus cell lines also suggest that themicronucleus plays a role in determining normal oralmembranellar pattern and length in the sexual cycle.

Heterospecific micronuclear transplantation, thetransplantation of a micronucleus of one species intoan amicronucleate of another species, offers an ad-ditional approach to the analysis of the stomatogenicfunction of the micronucleus. Micronuclearanomalies may develop in a foreign cytoplasm duringasexual and sexual reproduction, if incompatibilitybetween the two species exists, and the stomatogenicconsequences in such heterospecific transplants canbe studied. Furthermore, if the two species employeddiffer somewhat in their oral apparatus, the control ofthe micronucleus over particular stomatogenic stepscan be dissected in the heterospecific transplants.

In the present study, heterospecific micronucleustransplantation is performed using P. jenningsiamicronucleates as recipients and P. tetraurelia asmicronucleus donors. There is enough similarity be-tween the two species to warrant, a priori, thesuccessful maintenance and propagation of the micro-nucleus in the transplants. Both types of micronucleibelong to the 'aurelia' group (see Wichterman, 1986).They both undergo autogamy naturally upon star-vation and their micronuclear cycles in autogamy arelargely similar (for P. jenningsi, see Diller & Earl,1958; Mitchell, 1963). Furthermore, P. jenningsi

resembles P. tetraurelia in the control of the micronu-cleus over stomatogenesis, in both asexual and sexualreproduction (M.F.C. & S.F.N., in preparation; cf.Ng & Mikami, 1981; Ng & Newman, 19846). The oralapparatuses of the two species are similar in thepattern of the oral membranelles, but differ substan-tially in length (P. tetraurelia, 28fim; P. jenningsi,36/un) (Figsl, 2). There are thus both similaritiesand differences in the micronucleus-stomatogenesissystem in the two species that make them suitable forthe heterospecific analysis.

Homospecific transplantations, involving amicro-nucleate recipients and micronucleus donors of thesame species (P. tetraurelia), could generate geneti-cally normal cell lines (Ng, 1981) and rescue the cellsfrom stomatogenic abnormalities and failures in asex-ual and sexual reproduction (Ng & Tarn, 1987).Furthermore, in the Paramecium caudatum complex,heterospecific micronuclear transplantation betweentwo sibling species (syngens) has been shown to beable to promote recovery from growth depression(Fujishima & Watanabe, 1981). Heterospecific micro-nuclear transplantations are expected to entail differ-ent stomatogenic consequences, unless the micronu-clear stomatogenic signals in the two speciesconcerned have much in common.

Materials and methods

Cells and cultureTwo species of Paramecium were used (i) Parameciumtetraurelia stock d4- l l l , a derivative of stock 51 VII, ishomozygous for the nd3b gene and cannot discharge tricho-cysts when stimulated by saturated picric acid (for details,see Sonneborn, 1974). (ii) Paramecium jenningsi, obtainedfrom K. Hiwatashi, Japan, originally from the Sonneborncollection, USA. For culture and handling of cells, themethods of Sonneborn (1950, 1970) were followed. Phos-phate-buffered cerophyl medium (2-5 gl~ ' , pH7), inocu-lated with Enterobacter aerogenes and supplemented with5 m g r ' stigmasterol, was used. Experiments were per-formed at 27°C, while back cultures were stored at13-15-C.

Enucleation and renucleation of P. jenningsiEnucleation and renucleation were performed according tothe method described in Ng (1981), except one needle wasused. Four P. jenningsi amicronucleate cell lines (amic J l ,amic J2, amic J3a, amic J3b) were derived independentlyfrom three bimicronucleate clones no. 1-3 (amic J3a andJ3b being from the same clone, no. 3), by simultaneouslyremoving the two micTonuclei from a cell at the clonal agesof 18, 7, 30 and 30 fissions, respectively. At the same time,two unimicronucleate cell lines (mic J l , mic J2) were alsogenerated from bimicronucleate clones no. 1 and no. 2,respectively, by emicronucleation; a unimicronucleateclone, mic J3, was derived by isolation from clone no. 3

Interspecific nuclear transplantation in Paramecium 181

exhibiting mis-segregation of micronuclei during asexualpropagation.

Two schemes of renucleation experiments were per-formed: (i) Homospecific transplantation, with P.jenningsias micronucleus donors. Three renucleated cell lines, RJ2,RJ3a and RJ3b, were generated by renucleating amicro-nucleate cell lines, amic J2, J3a and J3b, at the clonal agesof 104, 45 and 50 fissions, respectively, (ii) Heterospecifictransplantation, with P. tetraurelia stock d4-lll as micronu-cleus donors. Eight renucleated cell lines were generatedfrom four amicronucleate cell lines, amic Jl, J2, J3a and J3b(RT1 from amic Jl , at 80 fissions; RT2.1 and RT2.2 fromamic J2, at 116 fissions; RT3a.l and RT3a.2 from amic J3a,at 48 fissions; RT3b.l, RT3b.2 and RT3b.3 from amic J3b,at 48, 54 and 54 fissions, respectively). The homospecifictransplants were collectively designated as RJ2-RJ3b, andthe heterospecific transplants as RTl-RT3b.3 in the text.The clonal relationship of these cell lines was apparent fromtheir listing in the tables.

Sampling and cytologyTo reveal oral and nuclear structures, cells in vegetativepropagation, and also postautogamous cultures in each cellline were sampled for silver impregnation (Chatton &Lwoff, 1936; Corliss, 1953) at different times (number offissions) after renucleation (for details, see Tables 1-4). Inthe sampling of postautogamous cultures, astomatous cellsof abnormal shapes were mostly not included. Samples ofthe progenitor cell lines, including P. jenningsi amicro-nucleate recipients, and P. jenningsi and P. tetraureliamicronucleate donors, were obtained at similar clonal agesfor comparison. The length of the oral apparatus wasdefined by the anteroposterior span of the quadrulus insilver-impregnated cells and measured with an ocular mi-crometer under x 1 000 phase-contrast optics (see Figs 1C,2B). For observing nuclei, aceto-orcein staining was alsoapplied (Beale & Jurand, 1966; without osmium fixation).

StatisticsOne-tailed Mest for comparison of mean length of the oralapparatuses with formula to cater for inequality of vari-ances and 2x2 G-test of independence for comparison oftwo percentages were performed as described by Sokal &Rohlf (1981).

Results

The maintenance of the micronucleus in asexualpropagation, nuclear reorganization in the sexualcycle, and the pattern and length of the oral appar-atuses in eleven renucleated cell lines (RJ2-RJ3bwith P. jenningsi micronuclei, RTl-RT3b.3 with P.tetraurelia micronuclei) in the asexual and sexualcycles are reported in the following sections.

Asexual reproduction(1) Maintenance of the micronucleus

Hie micronuclei in the renucleated cell lines did notexhibit any structural defects (Figs 3,4). However,

abnormality in the maintenance of the micronucleuswas evident in all renucleated cell lines, since theygave rise to amicronucleates. The percentage of cellspossessing micronuclei in the renucleated cell linesvaried from 25 % to 81 % (RJ2, 35 %; RJ3a, 53 %;RJ3b, 67%; RT1, 25%; RT2.1, 34%; RT2.2, 62%;RT3a.l, 81%; RT3a.2, 65%; RT3b.l, 57%;RT3b.2, 59%; RT3b.3, 78%; at 10-15 fissions afterrenucleation). This revealed the subnormal behav-iour of the transplanted P. jenningsi and P. tetra-urelia micronuclei during asexual propagation.Nevertheless, the micronucleus of both types couldbe maintained in the transplants for a long period: inmost of the renucleated cell lines, the percentage ofcells possessing micronuclei ranged from 55 to 72 %when assessed 35-48 fissions after transplantation;the three exceptions were RJ1 (0 %, 35 fissions), RT1(6%, 35 fissions) and RT2.1 (0%, 38 fissions).

(2) The pattern of oral membranellesAmicronucleates P. jenningsi experienced a de-pression period shortly after emicronucleation, dur-ing which the growth rate was reduced and the oralapparatuses were grossly abnormal in membranellarpattern and in length (Figs 5-7). However, theygradually recovered in growth rate, and the oralmembranellar pattern and oral length returned tonear normal in 30—60 fissions (to be detailed in aseparate communication).

In the present study, cell lines RJ2, RT1, RT2.1and RT2.2 were derived by renucleation of amicro-nucleates Jl and J2 that had already recovered (at62-116 fissions after enucleation, when only 2 % ofthe cells of amic J2 possessed slightly abnormal oralapparatuses, Table 1). These renucleated cell lines,as expected, also formed normal oral apparatusesafter renucleation (Table 1). However, all of theother transplants were derived by renucleation ofamicronucleates J3a and J3b that were still in thedepression period (at 15-24 fissions after enu-cleation). Interestingly, micronucleus reimplantationin these seven cell lines brought about a full recoveryin the pattern of oral membranelles in as early as10-15 fissions after renucleation, in contrast to theiramicronucleate progenitors which still exhibited sub-stantial percentages of abnormal oral apparatuses atthis time (Table 1, amic J3a, 23 %; amic J3b, 28 %).These amicronucleates had taken 15-30 fissions moreto approach full recovery in oral membranellar pat-tern (Table 1). This shows that micronucleus reim-plantation has brought about a faster recovery in oralmembranellar pattern. Significantly, the hetero-specific micronucleus (from P. tetraurelia) was ascompetent as the micronucleus from the same species(P. jenningsi) in promoting this recovery.


Figs 1, 4, 5, 6, 9 and 10 show the ventral view of silver-impregnated cells. Figs 3, 4, 8 and 9 show aceto-orcein stainingfor the nuclei. In all figures, the anterior end of the cell is oriented towards the top of the page. Magnification: Fig. 1,X1900; Figs 2, 5, 6, 10, 11, X1800; Figs 3, 4, 7, X1700; Figs 8, 9, X7000. Abbreviations: dp, dorsal peniculus; Iv, leftvestibule; ma, macronucleus; mal, macronuclear anlage; mi, micronucleus; oa, oral apparatus; pf, postoral fibres;q, quadrulus; vp, ventral peniculus.Figs 1, 2. Normal oral apparatuses of P. jenningsi (Fig. 1A,B,C, three focal levels) and P. tetraurelia (Fig. 2A,B, twofocal levels). Notice the similarity in their oral membranellar pattern: both consist of ventral and dorsal peniculi eachcomprising four closely packed basal body raws and four quadrular basal body rows widely spaced in the anterior part.At level X, the dorsal peniculus and quadrulus make a left turn and form an S-shape spiral while the ventral peniculusterminates. The length of the oral apparatus (dotted lines) of P. jenningsi is longer. The left vestibule and postoralfibres are also shown.Fig. 3. A vegetative cell from homospecific transplantation showing a typical P. jenningsi micronucleus.Fig. 4. A vegetative cell from heterospecific transplantation showing a P. tetraurelia micronucleus in P. jenningsicytoplasm. The P. tetraurelia micronucleus is vesicular and smaller than that in P. jenningsi.


J

Figs 5-7. Abnormal patterns of the oral membranelles in amicronucleate P. jenningsi. Fig. 5 shows the fragmentationof the posterior part of the ventral and dorsal peniculi at several sites (arrowheads). In Fig. 6 (A,B, two focal levels),the quadrulus is broken up into three portions with the middle portion (arrow) shifted to the (cell's) right while theanterior and posterior portions (arrowheads) are continuous with what appears to be an extra piece of peniculus in themiddle (ep). In Fig. 7 (A,B, two focal levels), the quadrulus and peniculi are broken up at different latitudes (arrows).The anterior and posterior penicular fragments are laterally displaced relative to each other. The buccal cavity isapparently compressed in an anteroposterior direction. This has caused the anterior fragment of the ventral peniculus toline up with the posterior fragment of the dorsal peniculus at a lower level (double arrow); additionally, the posteriorfragment of the quadrulus is 'pushed' anteriorly and bends where it is abutted by the posterior end of the anteriorfragment of the dorsal peniculus (arrowhead). The lines mark the length of the oral apparatus which is reduced.Fig. 8. A postautogamous cell of normal micronucleate P. jenningsi showing two typical macronuclear anlagen with'chromatinic centres' resembling bull's eyes, and one of the two micronuclei in focus.Fig. 9. A postautogamous cell from heterospecific transplantation showing one micronucleus and three homogeneousmacronuclear anlagen with morphology characteristic of P. tetraurelia.Figs 10, 11. Abnormal oral apparatuses in postautogamous cell of the renucleated cell lines. In Fig. 10, the ventral anddorsal peniculi and quadrulus are broken up into fragments that are laterally displaced. Only the middle fragment of thequadrulus is in focus (arrowhead). In Fig. 11, in the anterior part of the dorsal peniculus three basal body rows arespread out resembling the quadrulus, instead of being closely-packed (lines).


Table 1. Oral membranellar pattern of vegetative populations of amicronudeate cell lines (amic Jl-J3b) andrenucleated cell lines (RJ2-RJ3b, RTl-RT3b.3) at different times (numbers of fissions) after renucleationu

Cellline

amic JlRT1amic J2RJ2RT2.1RT2.2amic J3aRJ3aRT3a.lRT3a.2amic J3bRJ3bRT3b.lRT3b.2RT3b.3

Samplesize

946143513953624646596159506157

10-15:

%

fissions

of cells withb

Normal Abnormal1

OA

10010010094

10010076

10010010072

100100100100

OA

0006 n0 nnn0 nnn

23

o •••0 " *

o *••28

o •••o •*•0 ***

o •••

Samplesize

_—

48103962425232405047353552

16-30 fissions

% of cells with"

Normal AbnormalOA

—-

9810010010010010010010089

100100100100

OA

_-20 nn0 nn0 nn00 nnn0 nnn0 nnn

110 "0 '*0 "0 "

Samplesize

381241--24262741433045412835

31-50 fissions

% of cells withb

Normal Abnormal'OA

10010098--

1001001001001009799

100100100

OA

002

--

0 nn00 nnn0 nnn0 nnn31 nn0 nn0 nn0 nn

"Amicronucleate cell lines were sampled at similar ages to the renucleated cell lines.bOA, oral apparatus; abnormal OA, see text.C2x2 G-test of independence was performed for comparison of the % of cells possessing abnorrnal oral apparatuses between

renucleated cell lines and their amicronucleate progenitors: nnn, no difference; nn, 0-1 <P<0-9; n, 0 -05<P<01; *, 0-01 <P<005;•*,0-001<P<0-01; •'*, P<0-001.

(3) The length of the oral apparatusApart from the rapid recovery in membranellarpattern, renucleated cell lines from both homo- andheterospecific transplantations also exhibited aprominent increase in oral length compared toamicronucleates. In ten renucleated cell lines (apartfrom RJ2), the mean oral lengths of cells possessingmicronucleus in 26 vegetative samples (out of 28, atdifferent numbers of fissions after renucleation) weresignificantly longer than those of their amicronucleateprogenitors at similar ages (Table 2). Moreover, thehighest value of mean oral lengths exhibited bythe renucleated cell lines was also greater than thehighest value attained by the amicronucleate counter-parts. These observations demonstrate that themicronucleus participates in determining oral length,since its presence has allowed the development oflonger oral apparatuses approaching that in normalP. jenningsi (mean of mean oral length of the vari-ous renucleated cell lines = 34-4 /zm, range =30-0-37-3jtm, Table 2; c.f. normal oral length,Table 2, footnotes). The converse of the above obser-vations was exhibited by one renucleated cell line,RJ2; this could be due to abnormal functioning of thetransplanted micronucleus in this case.

As with the recovery of normal membranellarpattern, the recovery of oral lengths following micro-nucleus reimplantation was rapid. This is best shown

in the renucleated cell lines RJ3a, RJ3b andRT3a.l-RT3b.3 as these were renucleated withamicronucleates (J3a and J3b) within the depressionperiod. At 10-15 fissions after renucleation, theserenucleated cell lines already attained the mean orallengths of 32-2-35-9jim, while their amicronucleatecounterparts were only of 26-3-26-9/im. Their orallengths were even longer than those attained by theamicronucleates at 30 fissions later (Table 2, amicJ3a, 31-5 fim; amic J3b, 26-5^m).

Again, in both homo- and heterospecific trans-plants the increase in oral length was substantial. Theeffectiveness of the two types of micronucleus reim-plantation was analysed by comparing their mean orallengths (one-tailed f-test). To avoid interclonal vari-ations of oral length and also intraclonal variationsarising from different clonal ages, renucleated celllines belonging to the same clone (i.e. RJ3a vs RT3a.land RT3a.2; RJ3b vs RT3b.l, RT3b.2 and RT3b.3)and at similar ages were compared (Table 2, seefootnote c). In 4 of the 15 cases analysed, the meanoral length of the cell line renucleated with micronu-cleus from the same species was significantly greaterthan that in the heterospecific transplant; in 5 othercases there was no significant difference; the remain-ing 6 cases showed the converse. This ratio, 4:5:6, isessentially 1:1:1. There is thus no indication that theheterospecific micronucleus was different from the


Table 2. Mean length of the oral apparatuses of vegetative populations of amicronucleate cell lines (amic Jl—4) andrenucleated cell lines (RJ2-RJ3b, RTl-RT3b.3) at different times (number of fissions) after renucleation"

Cell

line

amic Jl

RT1

amic J2

RJ2

RT2.1

RT2.2

amic J3a

RJ3a

RT3a.l

RT3a.2

amic J3b

RJ3b

RT3b.l

RT3b.2

RT3b.3

Mean ± S . D .

(jim)

33-1 ±2-43

34-2 ±1-39

33-412-51

32-1 ±2-48

35-9 ±3-63

35-0 ±3-50

26-9 ± 3 1 2

33-0 ±2-26

33-1+2-10

34-6 ±1-7826-3 ±3-04

34-5 ±2-21

34-5 ±2-51

32 2 ± 2-89

35-9 ±1-81

10-15 fissions

(")

(94)

(61)

(43)

(51)

(39)(53)

(62)

(46)

(46)

(59)

(61)

(59)

(50)

(61)

(57)

Range

Oim)

27-3-36-8

31-5-36-8

25-2-37-8

27-3-35-7

24-2-38-9

26-3-38 9

16-8-33-6

27-3-37-8

28-4-37-8

30 0-37-8

17-9-31-5

27-3-38-9

28-4-38-9

27-3-36-8

31-5-38-9

Amicb

vs RJ& RT

• ••

( " )4 * *

• * •

* * *

* • •

* • •

• • *

• • •

* * *

RJC

vs

RT

nn

C)

nn*•

( " )

Mean ±s.D.

(jim)

33 1 ±2-19

300 ±2-06

33-1 ±3-43

33-9 ±2-68

29-2 ±2-45

35-4 ±1-97

33-9±3-14

33-1 ±2-47

26-2 ±3-36

34-0 ±1-79

32-8 ±1-95

33 8 ±1-45

35-3 ±1-61

16-30 fissions

(")

(48)

(10)

(39)

(62)

(42)

(52)

(32)

(40)

(50)

(47)

(35)

(35)(52)

Range

(jim)

-

-

25-2-37-8

26-3-32-6

27-3-38-9

28-4-38-9

26-3-33-6

30-0-38-9

29-4-38-9

27-3-37-8

17-9-33-6

30 5-36-8

27 3-36-8

30-5-36-8

32-6-38-9

Amicb

vs RJ

& RT

(•")nnn

*

#**

• ••

• **

***

• *•

*••

RJC

vsRT

*•

*•

nn

( " )

Mean ±S.D.

(jim)

32-112-76

33-2 ±2-03

30-614-17

34-511-53

31-5 ±2-09

33 3 ±2-18

35-812-03

34-711-81

26-512-10

34-611-53

34-311-41

35-011-52

37-311-44

31-50 fissions

(«)

(38)

(12)(41)

(24)

(26)

(27)(41)

(43)

(30)

(45)

(41)

(28)

(35)

Range

0"T>)

27-4-36-8

29-4-35-7

26-3-35 7-

-

31-5-37-8

27 3-36-8

27 3-35-7

30-0-38-9

31-5-38-9

23-1-30-5

30-5-36-8

30-5-36-8

30-5-37-8

33-6-38-9

Amich

vs RJ

& RT

n

***

• ••

• *•

• ••

• •*• *•

• • •

RJC

vs

RT

( " )( " )

nn

nn

( " )

°Same as footnote a in Table 1.b c One-tailed Mests comparing mean oral lengths of the cell lines. Probability values are represented by the same symbols as in Table 1 (see footnote c). In Amic vs RJ and

RT. bracketed symbols denote that the mean OA length of the amicronucleates is greater than that of the transplants. In RJ vs RT. bracketed symbols denote that the mean

OA length of the heterospecific transplants (RT) is greater than that of the homospecific transplants (RJ), RJ2 was excluded from comparison since it appeared to be

abnormal. Mean oral length of P. jenningsi micronucleate controls within 20 fissions after renucleation of the amicronucleates- mic J l , 37-411-54/an (n = 42); mic J2.

36-812-30f<m (n = 36); mic J3, 35-5 11-90/an (n = 51); these arc significantly greater than that of the renucleated cell lines belonging to the same clone, except RT3b 3

micronucleus of the same species in their stomato-genic function.

Because of postautogamous death in the homo-specific transplants (see below: section 2 of 'SexualReproduction'), the possibility exists that the trans-planted micronuclei were damaged during the oper-ation, probably due to their large size. This raises theconcern as to whether the stomatogenic functions ofthese micronuclei were impaired, and hence the orallengths attainable by homospecific transplantationmay be underestimated in the present study. Whilethis may well account for the situation in RJ2, webelieve that in RJ3a and RJ3b the micronuclearstomatogenic functions were largely normal, for thefollowing reason. In a previous study, homospecificmicronuclear transplants of P. tetraurelia maintainingnormal micronuclei (as demonstrated by good post-autogamous survival) nevertheless possessed oralapparatuses only of near-normal length (Ng & Tarn,1987). It was concluded that nonmicronuclear factorshad contributed to the subnormal oral length in thetransplants and that the micronuclear stomatogenicfunctions in such transplants were largely normal. Inthe present study, near-normal oral lengths wereattained in both homo- and heterospecific transplants(apart from RJ2). It thus seems to be the case that themicronuclei in these transplants were able to exercisenormal stomatogenic functions. In any case, it is

remarkable that a P. tetraurelia micronucleus canpromote near-normal recovery of oral length charac-teristic of P. jenningsi, in view of the large differencein oral length of the two species (P. tetraurelia, 28 fim;P. jenningsi, 36/xm).

Sexual reproduction(1) Nuclear reorganization

All of the renucleated -cell lines exhibited micronu-clear abnormalities during autogamy. This wascharacterized by the failure of some of the postautog-amous cells to possess either micronuclei or macronu-clear anlagen, with the latter situation being morefrequent, and others possessing none (Table 3, +,—and -,— classes). Even in cells possessing bothmicronuclei and macronuclear anlagen, some did notcontain the normal complement of two micronucleiand two macronuclear anlagen (2,2). Instead, theyexhibited 1,1; 3,3; 2,1; 3,1; 1,3 and rarely 4,4 nuclearconstitutions.

In the majority of cases analysed (10 out of 13), thepercentages of postautogamous cells exhibiting ab-normal nuclear constitutions in the heterospecifictransplants were significantly greater compared to thehomospecific transplants (Table 3; see footnote d fordetails). This shows that the heterospecific micronu-clei (from P. tetraurelia) behaved more abnormally in


Table 3. Oral and nuclear structures of postautogamous populations of renucleated cell lines (RJ2-RJ3b,RTl—RT3b.3) derived by autogamies induced at different times (numbers of fissions) after renucleation and of

micronucleate cell lines (mic J1-J3) at similar ages

Cellline

mic JlRT1

mic J2RJ2

RT2.1RT2.2

mic J3RJ3a

RT3a.l

RT3a,2

RJ3b

RT3b.l

RT3b2

RT3b.3

d4-lll

No. offissions

—

1335-1424111244-

1649124311421042124810371238_

Samplesize

73656048558250635222706171556068835675547143705029

+ ,+

1004956

1007182223214

1006389314

55506280592038286046

100

% of cells possessing'>b

Normal OA

+ , -

029200

167

4414190334

64127873

453

1814240

-,—

002096

3441460

333

65313219164

373149442022

0

Abnormal OA

+ , + •

08

16044002000001

12851265420

f , -

01260010340000006540000260

- , -

0200000

10150150106100245000

% of cells'possessing

abnormal OA

022220450 nn

13 n21 •*0150 nn1 nn1 nnn

24 "1491 (••)4 nn

10 nn10 nn6 n8 nn0

% of cells"of the + , - &

—,- classes

043280

251478 ***8 ***

84 " •0

371169 " •96 " •44 nn38 • "301540 nn78 • "56 " *67 " *36 nn52 • "0

"OA, oral apparatus; abnormal OA, see text.h + , + , with both micronuclei and macronuclear anlagen; +, —, either micronuclei or macronuclear anlagen were present with the

former being more frequent; —, —, no micronuclear derivatives.C2x2 G-test was performed to assess if the % abnormal OA in heterospecific transplants was significantly greater than that in

homospecific transplants. Comparisons were made between postautogamus populations derived by autogamies induced at equivalentnumbers of fissions after renucleation from the two types of renucleated cell lines, both being originated from the same amicronucleatecell line in each case. For example, RT2.1 at 11 fissions and RT2.2 at 12 fissions were compared to RJ2 at 14 fissions; RT2.2 at 44fissions was compared to RJ2 at 24 fissions. The probability values are represented by the same symbols as in Table 1 (see footnote c).Where the symbol is bracketed, it denotes that the % abnormal OA in heterospecific transplants was significantly smaller than that inhomospecific transplants.

d2x2 G-test was performed to assess if the % abnormal nucleogenesis in heterospecific transplants was significantly greater than thatin homospecific transplants. The condition of comparison and symbols are as in footnote c.

nucleogenesis compared to the homospecific ones(from P. jenningsi) in the cytoplasm of P. jenningsi.

The macronuclear anlagen formed in the homo-and heterospecific transplants were morphologicallydifferent and conformed to their specific origins. Inthe former, the anlagen were characterized by thepresence of 'chromatinic centres' resembling bulleyes (Fig. 8), typical of P. jenningsi (Diller & Earl,1958; Mitchell, 1963). The latter had homogeneousanlagen resembling that of the micronucleus donor,P. tetraurelia (Fig. 9). Both types of anlagen in thetransplants failed to support somatic functions result-ing in 100 % postautogamous death.

(2) The initiation of oral membranelle assemblyWe have shown previously in P. tetraurelia that,during sexual reproduction, the micronucleusmediates an early stomatogenic step of oral membra-nelle assembly ('initiation'); in amicronucleates, sto-matogenesis becomes arrested at this crucial stepresulting in astomy and death (see Introduction). Thisunique stomatogenic function of the micronucleuswas also demonstrated by amicronucleate P. jenn-ingsi cell lines in autogamy (to be detailed separatelyin another report). The importance of the presence ofthe micronucleus in the renucleated cell lines wasfurther demonstrated by isolating, from renucleatedcell line RT1, five vegetative cells that had lost the


renucleated micronuclei and expanding these into celllines; they also failed to initiate oral membranelleassembly and became astomatous after autogamy(total 227 cells sampled). In contrast, in sublines ofboth homo- and heterospecific micronuclear trans-plants maintaining micronuclei the development of anew oral apparatus was possible in some, though notall of the autogamous cells, so that the postautog-amous culture contained feeders, besides the thin,deformed astomatous nonfeeders. This shows thatthe presence of the micronucleus, even of anotherspecies, can mediate the initiation of oral membra-nelle assembly and the subsequent development ofthe oral apparatus. Clearly, the micronuclear signalfor this crucial stomatogenic step is not species-specific.

The astomatous postautogamous cells of the trans-plants had originated from two sources. As notedabove, the renucleated cell lines generated someamicronucleates during asexual propagation. Obvi-ously, some of the postautogamous astomatous cellswere derived from such amicronucleates present inthepreautogamous culture. Others were generated asa result of abnormal nucleogenesis in micronucleatesduring autogamy, in particular when the micronu-clear cycle had become arrested after meiosis so thatnone of the postmeiotic micronuclear derivativeswere retained for further nuclear development (seeTarn & Ng, 1986; Chau & Ng, 1988a). Because of theheterogeneous origin of the astomatous postautog-amous cells, the exact percentage of the latter typewas not assessed in the present study. Inspection ofthe postautogamous cultures of the heterospecifictransplants revealed that up to 50 % of the cells werefeeders in some cases. This indicates that in a signifi-cant proportion of the autogamous cells of the hetero-specific transplants, the micronucleus was competentin mediating the initiation of oral membranelle as-sembly. This ability, however, was not compared inthe homo- and heterospecific transplants.

The postautogamous feeders were selectively har-vested for silver impregnation, to allow a comparisonbetween the homo- and heterospecific transplants intheir oral apparatus and in nucleogenesis (see sec-tions 3 and 4 below).

Some postautogamous cells possessing oral appar-atus nevertheless did not bear any micronuclei ormacronuclear anlagen (Table 3, —,- cases). It islikely that, in these, the micronucleus had exercisedits stomatogenic function (in the initiation of oralmembranelle assembly) before disappearing later inthe nucleogenic cycle. This aspect has been exploredand discussed in some detail in another study employ-ing P. tetraurelia cell lines bearing defective micronu-clei (Chau & Ng, 19886, and unpublished data).

(3) The pattern of oral membranellesThe new oral apparatuses formed in the autogamousrenucleated cell lines were not always normal(Table 3). The two types of transplants gave rise tosimilar types of oral abnormalities, including frag-mentation of oral membranelles (involving the quad-rulus, but more often also dorsal and ventral peniculi;Fig. 10), spreading out of the anterior basal bodyrows of the dorsal peniculus (Fig. 11), and reductionof the length of oral membranelles and buccal cavity.These oral defects were similar to those exhibited bycell lines possessing defective micronuclei inautogamy (Tarn & Ng, 1986; Chau & Ng, 1988aAand unpublished data), suggesting that the oral ab-normalities in the renucleated cell lines had alsostemmed from abnormal micronuclear functioning inthe sexual cycle.

Unlike nuclear reorganization, postautogamouscells from the homo- and heterospecific transplantsexhibited similar degrees of oral membranellar ab-normalities. When the percentages of postautog-amous cells possessing defective oral apparatuses inthe two types of transplants from the same clone werecompared, no significant difference was found in tencases (Table 3, see footnote c for details). In twoother cases, the heterospecific transplants showedsignificantly higher percentages of oral abnormalities,while the remaining one case showed the converse. Inaddition, there was probably only a slight indication,if any, of more oral abnormalities in the hetero-specific transplants, in which the highest percentageof oral abnormalities was greater than that'observedin the homospecific transplants (RT3a.2, 24% vsRJ3b, 14%). This analysis therefore shows thatwhenever an oral apparatus was formed in the trans-plants in autogamy, the probability of producing anabnormal oral membranellar pattern and the types ofabnormalities produced were about the same in thetwo types of transplants. This suggests that themicronuclear stomatogenic signals in the patterningof the oral membranelles during sexual reproductionare largely similar in the two species.

(4) The length of the oral apparatusThe oral apparatuses formed in autogamy in therenucleated cell lines were mostly significantly longerthan that of P. tetraurelia (14 out of 15 cases of theheterospecific transplants), but shorter compared tothe micronucleate P. jenningsi controls (13 out of 21cases of homo- and heterospecific transplants,Table 4). As in asexual reproduction, the homo- andheterospecific transplants produced oral apparatusesof similar lengths during autogamy (Table 4). Out of13 cases compared, in 4 cases the oral lengths attainedby the homospecific transplants was significantlygreater. In another 4 cases, there was no significant


Table 4. Mean length of the oral apparatuses of postautogamous populations of renucleated cell lines (RJ2-RJ3b, RTl-RT3b.3) derived by autogamies induced at different numbers of fissions after renucleation and of

micronucleate cell lines (mic J1-J3) at similar ages

Cellline

mic JlRT1

mic J2RJ2

RT2.1RT2.2

mic J3RJ3a

RT3a.l

RT3a.2

RJ3b

RT3b.l

RT3b.2

RT3b.3

No. offissions

_

1335-1424111244-1649124311421042124810371238

P. tetraurelia

Mean± S.D.On)

36-5 ±1-5932-0 ± 1-4132-4 ±3-2735-9 ± 1-9234-8 ±2-3631-4±3-8631-2 ±2-4531-7±3-2533-2 ±2-9532-7 ±1-8632-6 ±2-7131-8±3-1332-7 ± 1-9132-8 ±2-3031-9 ±2-4529-7 ±2-9629-3 ±3-7632-5 ±2-5931-4 ±2-8432-8 ± 1-8728-5 ±3-6832-2 ± 2-0332-4 ±2-6434-0 ±2-56

28-9+ 1-31

(")

(73)(65)(60)(48)(55)(82)(50)(57)(44)(65)a

(70)(61)(71)(55)(60)(62)(73)(56)(75)(54)(71)(43)(70)(50)

(29)

RangeOm)

32-6-38-925-2-36-826-3-36-831-5-38-930-5-38-921-0-36-828-4-37-822-1-36-826-3-38-929-5-36-826-3-36-823-1-36-828-4-36-826-3-36-725-2-36-823-1-35-721-0-34-727-3-36-821-0-36-827-3-36-825-2-34-726-3-35-721-2-36-830-5-36-8

26-3-30-5

Comparison of mean lengths

with P. jenningsimicronucleate

cell lines

_• • •

-

• •

• • *

* * *

* * *

-

nn*

nnnnn*

***• ••nn• • •

nn***nn

( " )

with P. tetraurehamicronucleate

donors

_• • *

• • *

-

-

—

• • •

* * *

* * *

-

-

-

• **

• • *

* * *

*

-

-

* * •

• * •

nn** *** •**•

b

between homo- &heterospecific trans-plants in each clone

_-----

******

C")--_nn

( " )*•**••--

("*)nnn

nn

(*")( " )

'Two samples were pooled in which no significant difference was found.b One-tailed (-test. Comparisons of homo- and heterospecific transplants were made with samples at similar numbers of fissions after

renucleation; probability values are symbolized (see footnote c in Table 1 for details). Probability values enclosed in brackets indicatethat the mean oral length in the heterospecific transplant was significantly greater.

difference between the two. The remaining 5 casesdemonstrated the converse. This ratio, 4:4:5, isobviously not different from 1:1:1. Thus, the hetero-specific micronucleus was as effective as the homo-specific one in the provision for the development ofnormal oral length. It is of interest to emphasize thatthe oral length exhibited by the heterospecific trans-plants were mostly significantly longer than that ofthe P. tetraurelia micronucleus donors, and ap-proaching that of the recipient species, P. jenningsi(Table 4, mean of mean oral length of the cell linesfrom heterospecific transplantation, 32-0//m, range,28-5-34-

Discussion

In the present study, homo- and heterospecific micro-nuclear transplantations entailed similar stomato-genic consequences in asexual as well as sexualreproduction. During vegetative propagation, the

heterospecific transplants were as competent as thehomospecific transplants in effecting recovery ofthe membranellar pattern to normal and in allowingthe development of longer oral apparatuses withlength approaching that of micronucleate P. jenn-ingsi. Moreover, the recovery in both types of trans-plants was rapid, taking only 10-15 fissions.

During autogamy, micronuclei from P. tetraureliacould trigger the initiation of oral membranelle as-sembly and consequently the formation of functionaloral apparatuses in the heterospecific transplants. Asin asexual reproduction, the oral lengths attained bythe heterospecific micronuclear transplants were notsignificantly different from those reimplanted withmicronuclei from the same species and, remarkably,they approached the oral length of the cytoplasmicrecipient (P. jenningsi) instead of the micronucleatedonor (P. tetraurelia). In addition, similar percent-ages and types of oral membranellar abnormalities inpostautogamous cells were demonstrated by the


homo- and heterospecific transplants. These obser-vations allowed the following conclusions on micro-nuclear stomatogenic functions to be made.

Conserved micronuclear stomatogenic functionsThe development of an oral apparatus during vegetat-ive propagation and autogamy can be subdivided intotwo stages: the critical step of initiation of oralmembranelle assembly ("initiation") and subsequentstomatogenic developments. In previous studies, themicronucleus was shown to exercise control over theinitiation of oral membranelle assembly during sexualbut not asexual reproduction, and it also affected thepatterning of the oral apparatus during both cycles(see Introduction).

The ability of a heterospecific micronucleus totrigger oral membranelle assembly during sexualreproduction in the cytoplasm of a different species isparticularly revealing. This clearly indicates that themicronuclear stomatogenic controls over the in-itiation step in the two Paramecium species havemuch in common. The similarity of this micronuclearstomatogenic function in the two species suggests thatthis micronuclear function is fundamental to thegenus.

The other micronuclear stomatogenic functions, inthe control of oral membranellar pattern during celldivision, are also conserved. Both homo- and hetero-specific micronuclear transplantations conferredequally rapid recovery of membranellar pattern tonear-normal condition, within 10-15 fissions of pro-pagation of the transplants. Additionally, micronu-clear control over the patterning of the oral apparatusduring the sexual cycle can also be interpreted asconserved, since the percentages and types of oralmembranellar abnormalities exhibited by postautog-amous cells from homo- and heterospecific trans-plants were similar. Understandably, this last obser-vation is open to other explanations, since similarityin oral abnormality in the sexual cycle in the two typesof transplants does not necessarily imply similarmodes of micronuclear controls in them. The com-parison of the nature of micronuclear controls on oralpatterning during autogamy in the two species there-fore awaits further investigation.

Micronuclear function in the determination of orallengthThe micronucleus also plays a role in determining thelength of the oral apparatus during asexual and sexualreproduction (see Introduction). Heterospecificmicronuclear transplantation is particularly illuminat-ing in this regard. The micronucleus donor, P.tetraurelia, has a much shorter oral apparatus, com-pared to that of the recipient, P. jenningsi (28 vs36fxm). The heterospecific transplants thus furnished

a test of the efficacy of micronuclear control over orallength.

Both homo- and heterospecific transplants devel-oped oral apparatuses comparable in length to that ofthe cytoplasmic recipient P. jenningsi, in asexual andsexual cycles. Hence, the function of the micronu-cleus in the determination of oral length is notspecies-specific. Whereas the micronucleus providesthe necessary conditions for the development ofnormal oral length, the oral length characteristic of aspecies is determined by nonmicronuclear factors. Amacronucleus-acting gene, shl, has been shown to beinvolved in the determination of oral length in P.tetraurelia (Tarn & Ng, 1987).

Incompatibility between P. tetraurelia micronucleusand P. jenningsi cytoplasmIn the studies of Ng (1981) and Tarn & Ng (1987)employing P. tetraurelia, about two thirds of cell linesgenerated by homospecific micronuclear transplan-tation were normal in the propagation of the micro-nucleus during cell division. In addition, nuclearreorganization during the sexual cycle was also mostlynormal, as shown by the high survival rate andnormal expression of specific genes derived from theimplanted micronuclei. However, in the presentstudy, all of the homo- and heterospecific renucleatedcell lines were abnormal in micronuclear propagationduring vegetative division and in nuclear reorganiz-ation during sexual reproduction. All postautog-amous cells of these transplants died, except whenthere was regeneration of fragments of the recipients'presexual macronuclei. Could these micronuclearabnormalities be the result of damage of the micronu-clei during transplantation? While this could well bethe reason in the homospecific transplants, because ofthe large size of the P. jenningsi micronucleus, we donot think this accounts for the situation in theheterospecific transplants, as the size of the P.tetraurelia micronucleus is much smaller. Moreover,the heterospecific transplants exhibited a higher per-centage of abnormalities in nuclear reorganization inthe sexual cycle, suggesting interspecific incompati-bility between the micronucleus and the recipient. Ithas been well documented that the micronucleuscomes under the influence of the cytoplasm duringnuclear reorganization in different phases of thesexual cycle: the progress through premeiotic S phase(Fujishima & Hiwatashi, 1978); the survival of onepostmeiotic product in the paroral cone (Sonneborn,1954; Yanagi & Hiwatashi, 1985); the development ofthe zygotic nucleus (Harumoto & Hiwatashi, 1982)and the differentiation of macronuclear anlagen(Mikami, 1980; Grandchamp & Beisson, 1981; Mik-ami & Ng, 1983). Moreover, incompatibility has alsobeen shown in interspecific conjugation between


sibling species of the Paramecium aurelia complex(see Sonneborn, 1974), and also in interspecificnuclear transplantations in Amoeba and in amphib-ians (reviewed by Jeon & Lorch, 1979; Gallien, 1979).

An analogy with inductive interactions inmulticellular systems

The present finding is analogous to heterospecific(e.g. chick-duck; chick-mouse) dermal-epidermalinductive interactions in the development of feathersand hairs (reviewed by Sengel, 1971; Deuchar, 1975).The mesodermal inductive stimulus on the differen-tiation of epidermal feather or hair was shown to becommon to both species, in addition to the involve-ment of other species-specific factors. Similarly, theParamecium micronucleus exerts an 'intracellularinductive stimulus' common to the two species on theoral anarchic field to promote oral development, butthe final length of the oral apparatus is determined byspecies-specific factors residing outside the micronu-cleus. This analogy is particularly interesting in viewof the close spatial relationship between the oralanarchic field and the micronuclear postmeiotic div-isional derivatives at the time when the latter aresupposed to execute their stomatogenic functions(see Introduction).

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(Accepted 4 February 1988)

Documents

Interspecific micronuclear transplantation in Paramecium ...Interspecific micronuclear transplantation in Paramecium: nucleogenesis and stomatogenesis in asexual and sexual reproduction