Abortive Conjugation Induced by UV-B Irradiationat Meiotic Prophase in Tetrahymena thermophilaTAKASHI KOBAYASHI AND HIROSHI ENDOH*Department of Biology, Faculty of Science, Kanazawa University, Kanazawa, Japan

ABSTRACT Conjugating Tetrahymena were irra-diated by ultraviolet-B (UV-B) at various stages of conjuga-tion. When the conjugants were exposed to the UV-B atlate meiotic prophase (the stage from pachytene todiplotene), abortive conjugation was induced at highfrequencies. After completing meiosis, a significant num-ber of the conjugants showed marked anomalies, i.e.,failure of nuclear selection after meiosis, and abortion ofthe subsequent conjugation process such as a postmei-otic division to form gametic nuclei, nuclear exchange,synkaryon formation, and postzygotic development. Theconjugating pairs retained the parental macronucleusand separated earlier as compared with a control. Theresultant exconjugants degenerated meiotic products andbecame amicronucleates. These observations strongly sug-gest the presence of a UV-sensitive molecule that isexpressed specifically at the meiotic prophase and thatdirects the subsequent development after meiosis. Dev.Genet. 23:151–157, 1998. r 1998 Wiley-Liss, Inc.

Key words: UV-B; meiotic prophase; Tetrahymenathermophila; abortive conjugation

INTRODUCTIONUltraviolet-B (UV-B)(the wavelength range 280–320

nm) is known to be harmful to various biologicalmaterials and functions. The UV range induces DNAdamage, which may then be subject to repair [reviewedby Friedberg, 1985]. It also inactivates various mol-ecules, such as RNA, amino acids, protein, and virus. Insome instances of eukaryotes, UV causes swelling andvacuolation of mitochondria in Smittia [Kalthoff et al.,1975] and in Xenopus [Ikenishi et al., 1974]. In certaincases, a specific cell function is lost due to the UV-induced destruction of materials, one of which might bea key gene product. For example, pole cell formation inthe early development of Drosophila was inhibited byUV irradiation [Geigy, 1931; Jura, 1964; Okada et al.,1974]. The target molecule was found to be a maternalRNA that functions as a determinant of pole cellformation [Okada and Kobayashi, 1987]. Thus UVirradiation is considered a useful tool for providinginsight into various biological mechanisms such asciliate conjugation.

Conjugation of the ciliated protozoan Tetrahymenathermophila consists of the following cascade of events:meiosis, postmeiotic division to produce gametic nuclei,nuclear exchange, fertilization, postzygotic divisions,nuclear differentiation into somatic macro- and germi-nal micronuclei, and resorption of the old macronucleus[reviewed by Orias, 1986]. Abnormal forms of conjuga-tion are useful to dissect the normal conjugation se-quence, even though they are induced spontaneously orartificially. A conspicuously aberrant form of conjuga-tion is referred to as a genomic exclusion and has beenrepeatedly documented and analyzed [Allen, 1963; 1967;Nanney and Nagel, 1964; Doerder and Shabathura,1980; Gaetrig and Kaczanowski, 1987]. When a normalclone mates with an ‘‘aged’’ clone with a defectivemicronucleus, the micronuclear genome of a member ofa conjugating pair is completely excluded from theprogeny. The micronucleus-defective partner of the pairfails to produce functional gametic nuclei, whereas theremaining partner cell goes through normal processand produces two gametic nuclei. After unilateral trans-fer of one gametic nucleus from the normal to thedefective partner, the subsequent development isaborted, and the parental macronucleus is retained.This phenomenon appears to be the exclusive pathwayof conjugation in aging lines that have large micro-nuclear defects or hypodiploidy. Thus, analyses of ge-nomic exclusion as a form of abnormal mating havecontributed to the understanding of the conjugationpathway.

In the ciliated protozoa, various UV effects have beenreported, such as killing, inhibition of cell division, anddefective repair system [Calkins, 1963; Calkins et al.,1987; Yamamoto et al., 1997]. However, the effect on theconjugation process has not been fully analyzed. In theexperiment described in this paper, cells of Tetrahy-mena thermophila at various stages of conjugationwere irradiated by UV-B; thereafter, their conjugationprocess was monitored. In this work, we noted a

*Correspondence to: Hiroshi Endoh, Department of Biology of GeneralEducation Hall, Kanazawa University, Kanazawa 920–1192, Japan.E-mail: hendoh@kenroku.kanazawa-u.ac.jp

Received 21 April 1998; Accepted 13 July 1998

DEVELOPMENTAL GENETICS 23:151–157 (1998)

r 1998 WILEY-LISS, INC.

distinctly sensitive period to the UV-B at late meioticprophase (stages IV and V) and that the irradiationinduces nuclear anomalies similar to, but somewhatdifferent from, other aberrant forms of conjugation. Wealso discuss the relationship between the UV-inducedabortive conjugation and a UV-sensitive molecule.

MATERIALS AND METHODS

Stocks

Strains of Tetrahymena thermophila used in thisstudy were WA6 and WD6, kindly supplied by T. Sugai(Ibaraki University, Ibaraki, Japan).

Culture Methods and Induction of Conjugation

Conditions for cell culture, starvation, and conjuga-tion were followed by Sugai’s methods (personal commu-nication; Haremaki et al. [1996]). Cells were grown in0.25% proteose peptone, 0.25% yeast extract, and 3.5%glucose at 26°C. When the culture reached the late logphase, cells were washed three times with a starvationsolution (0.5 3 NKC: 17 mM NaCl, 0.5 mM KCl, 0.5 mMCaCl2) and resuspended in the same solution. Celldensity was adjusted to 1.0 3 105 cells/ml in a glassPetri dish, and the cells were incubated overnight at26°C. To induce conjugation, equal numbers of bothstrains were mixed and kept at 26°C.

UV Irradiation and Observation

Conjugants were exposed to UV-B (wavelength range280–320 nm) under a UV lamp FL20S-E (Toshiba). Toremove UV-C ,280 nm, the UV lamp was covered witha cellulose acetate sheet (Daicel Craft Ltd.). Because itwas confirmed that the survival and the followingproliferation of the cells in vegetative phase were notseriously affected by UV-B irradiation within 5 min,cells were exposed to the UV-B (1.72 W/m2/sec) for 5 minin all our experiments. The average power densityincident on the sample is approximately 516 W/m2.After irradiation, the cells were kept at 26°C and thenfixed with formalin (final 3%). The fixed cells werestained with 1 µg/ml DAPI (4,6-diamidino-2-phenylin-dole dihydrochloride) and observed by fluorescencemicroscopy.

RESULTS

Dose Determination of UV-B

As is well known, UV-C has a strong, harmful effect.In our preliminary experiments, UV of wavelength,280 nm (UV-C) was used. Indeed, UV-C produced adeleterious effect on cell growth after a short irradia-tion, ultimately leading to cell death. Therefore, weused UV-B in this experiment. UV from the lamp wasfiltered through a cellulose acetate sheet, and most ofthe UV-C component was removed. To determine theadequate dose of UV-B, vegetative cells were exposed

for 1–60 min; they were then isolated in culture me-dium. After 2 days, growing cells were counted as asurvival (Table 1). As the irradiation time was elon-gated, the survival ratio gradually decreased. Whencells were exposed to UV-B for within 10 min, morethan 90% of cells isolated grew vigorously at the samegrowing rate as that of unirradiated cells. Cells exposedfor 30 min initially showed little slow growth, but theysoon recovered normal growth rate. Although cellsexposed for 60 min showed more than 65% survivalratio after 2 days, it appears that the cells suffered fromserious lesions, because most of the isolated cell linesshowed extremely slow growth rate, and some portionof the cells stopped growing after several fissions. Fromthese results, we judged the 5-min exposure time not tobe harmful, at least to the vegetative growth. Thus, thefollowing study was carried out under these conditions.

Nuclear Events in UV-Induced AbortiveConjugation

Conjugating cells were exposed every one hour aftermixing cells of complementary mating types and keptat 26°C until 12, 15, and 20 h after mixing. Theexconjugant cells were then stained and observed todetermine whether conjugation proceeded normally. Innormal conjugation, at 12 or 15 h after mixing, cells areusually in the stage of macronuclear development,where they have two anlagen, one old macronucleusand two micronuclei. When cells were irradiated at 4and 5 h after mixing, remarkable abnormalities innuclear events were detected, as shown in Figure 1.Cells that developed macronuclear anlagen drasticallydecreased in frequency. They had only one old macro-nucleus and several pyknotic micronuclei (judging fromtheir sizes). During this highly UV-sensitive period ofconjugation, the cells were found to be predominantlyat stage IV (pachytene) or V (diplotene) (Fig. 2). Theabove results suggest that at the late meiotic prophase

TABLE 1. Percentage of Growing Cells AfterUV-B Irradiation*

Duration time forUV exposure (min)

% of growing cellsafter 2 days

0 1001 1005 97.1

10 91.530 77.2a

60 65.7b

*Cells were washed in 1/2 NKC 1 day before irradiation. Afterirradiation, at least 46 cells were isolated in culture mediumand kept at 25°C. Although the growth was observed until 3days after, the cells exposed for 1–10 min fully grew after 2days.aCells after isolation showed initially a little slow growth rate,but no difference from control cells was detected after 2 days.bMost isolated cell lines showed extremely slow growth rate orstopped growing after a few fissions.

152 KOBAYASHI AND ENDOH

there is a distinctly sensitive period for conjugatingcells to UV-B, and that a UV-sensitive molecule thatexists at the stage might be directing the followingprocess of conjugation.

In an effort to determine at which point the irradi-ated cells aborted conjugation, we focused on the nuclearbehavior after the UV irradiation at 5 h after mixing. Inthe irradiated cells, meiosis proceeded normally, result-ing in a generation of four haploid meiotic products.The cells, however, failed the nuclear selection aftermeiosis. It was observed that four meiotic productsdispersed in a cell. None of them attached to thejunctional zone (the cortex in the vicinity of paroralzone) (Fig. 3A). Thereafter, they moved to the posteriorend of the cell, where they finally became pyknotic at 9h after mixing (Fig. 3B). The conjugating cells did notundergo the subsequent postmeiotic division to producemigratory and stationary nuclei prior to nuclear ex-change. In addition, in rare cases, deformed macro-nucleus attached to the junctional zone as if it had beenone of the surviving haploid micronuclei (data notshown). In the most pairs, none of the expected pro-cesses was observed including nuclear exchange, fertil-ization, postzygotic divisions, macronuclear differentia-tion, and absorption of the parental macronucleus.Only several degenerating micronuclei were visible inexconjugants 12 h after mixing (Fig. 3C). The exconju-gants then lost all the micronuclei and finally died (Fig.3D). They transiently reconjugated as in the round 2

pairs (Bruns, 1986), but the pairs separated in amoment probably because of their amicronucleate state(data not shown). The pair separation started earlierthan the unirradiated control. For example, more than60% of the irradiated pairs had already separated after

Fig. 1. Frequency of exconjugants in which macronuclear anlagendeveloped 15 h after mixing of complementary mating types. Cellswere exposed every hour after mixing. The cells were stained withDAPI and observed 15 h after mixing. Irradiation at 4 and 5 h waseffective in aborting macronuclear development. Cells 5 h after mixing(stage V, diplotene) were most sensitive to the UV-B.

Fig. 2. Frequency of cells at different stages of conjugation 3–6 hafter mixing. A: At 3 h after mixing. Cells in stage II are predominant.B: At 4 h. Most of conjugants entered stage IV (pachytene) but noconjugant in stage V (diplotene). C: At 5 h. Conjugants were predomi-nantly at stage V. D: At 6 h. Most conjugants already completedmeiosis II. I–VI, meiotic prophase [Sugai and Hiwatashi, 1974]; M1,meiosis I; M2, meiosis II; PM, postmeiotic stages.

UV-INDUCED ABORTIVE CONJUGATION IN TETRAHYMENA 153

12 h, whereas the unirradiated control pairs alwaysshowed only less than 1%. Unirradiated controls at 9 hand 15 h after mixing are shown in Figure 3E and 3F,respectively. These cells showed the typical nucleardeveloping macronuclei and diploid micronuclei areobserved.

These observations show that conjugation was blockedafter completion of meiosis and the subsequent develop-ment was all aborted. The exconjugants retained theirparental macronucleus, but the meiotic products degen-erated without endoreplicating DNA, different from theround 1 genomic exclusion.

Efficiencies of Abnormal Nuclear Events

The above-mentioned anomalies were induced athigh frequencies. Figure 4 shows the frequencies of thecells in which the normal and abnormal nuclear eventsoccured 12, 15, and 20 h after mixing. The frequency ofthe cells in which normal development occurred wasless than 10%. The frequency of cells with the pyknoticmicronuclei decreased with the lapse of time; at thesame time, the percentage of amicronucleates in-creased gradually. The frequency of amicronucleates at20 h after mixing reached more than 65%. When

Fig. 3. Nuclear events after the UV irradiation. Conjugants wereexposed to UV-B 5 h after mixing complementary mating types. A: At 7h after mixing. The irradiated cells underwent meiosis, resulting ingeneration of four haploid meiotic products. However, the cells failednuclear selection after meiosis. Four meiotic products dispersed in acell (the cell on the right side in the pair) and none of them attached tothe paroral zone. B: At 9 h. The meiotic products moved to theposterior end of the cell, where two have already become pyknotic (thecell on the left side in the pair). C: At 15 h. Four degenerating

micronuclei were still visible in the exconjugant. D: At 24 h. Exconju-gants became amicronucleate and finally died. E: Unirradiated controlat 9 h after mixing. The conjugant has a typical configuration of nuclei,one old macronucleus, two faintly stained macronuclear anlagen anddiploid micronuclei. F: Unirradiated control at 15 h. The exconjugantstill keeps the same three types of nuclei as those in E, but absorptionof the old macronucleus has already begun. OM, old macronucleus; A,macronuclear anlage; m, micronucleus; p, pyknotic micronucleus.

154 KOBAYASHI AND ENDOH

summed up, the cells with pyknotic micronuclei and theamicronucleates whose frequencies aborted conjuga-tion reached more than 80%. These results suggest thatUV irradiation at meiotic prophase effectively inducesthe abortive conjugation, resulting in the amicronucle-ate state in exconjugants. Although the other types ofabnormality were also observed in a small portion ofexconjugants in which the number of anlagen wasbiased, such cells did not abort conjugation in that theydeveloped anlagen. These results suggest that the mostsensitive molecule to UV exists at meiotic prophase,although UV might affect several kinds of moleculeswhich play a role in different stages of conjugation.

DISCUSSIONWhen the conjugating cells received UV-B irradiation

at the late meiotic prophase (stage IV and V), asignificant number of the conjugants aborted the conju-gation process after meiosis. The results described inthe present paper suggest that a key factor such as aspecific gene product that would be synthesized andmaintained at the meiotic prophase might have a rolein regulating the subsequent developmental processafter meiosis in conjugation. The small dose of UV usedin our experiments did not affect the growth of vegeta-tive cells. Most UV-sensitive molecular species might bebarely synthesized. Such molecules at a particular

stage in conjugation must easily suffer degradation byUV, compared with other abundant molecules. Whattype of molecule was damaged by UV-B irradiation inthis experiment? We discuss this problem from mainlytwo points of view.

Micronuclear DNA at Meiotic Prophaseas a Possible Target Molecule

It is known that recombination occurs during the firstmeiotic prophase. In particular, zygotene and pachy-tene are organized to sustain the events required forrecombination between homologous chromosomes. Mostof the programmed nickings, gappings, and repairsyntheses occur during pachytene [Stern and Hotta,1984]. Considering these facts, chromosomal DNA maybe susceptible to breakage by UV at the stage ofrecombination. Micronuclear DNA at the meiotic pro-phase in Tetrahymena may also be particularly sensi-tive to UV. This view leads to the idea that theUV-damaged molecule is micronuclear DNA and theresultant micronuclear defect is responsible for theabortive conjugation as in genomic exclusion, althoughthe micronuclei after irradiation remain morphologi-cally normal until they become pyknotic.

With reference to micronuclear defect, Ward et al.[1995] showed that exconjugants derived from cellswith a wild-type macronucleus and nullisomic micro-nucleus fail to complete pair separation, to eliminateone micronucleus, and to amplify anlagen DNA. Theseabnormalities occur in the later stage of conjugationafter macronuclear differentiation. In the nullisomicstrain, nuclear selection and some subsequent steps inpostzygotic development proceeded normally. This nul-lisomic strain might lack several genes that are ex-pressed in the newly developed anlagen and are in-volved in these abnormalities, but might preservegenes which are expressed at meiotic prophase anddirect the earlier process of conjugation. In this respect,this deficiency is fundamentally different from that inthe UV-induced abortive conjugation and genomic exclu-sion. A micronuclear defect such as this nullisomy doesnot necessarily cause the serious abnormality. As men-tioned previously, genomic exclusion does occur whencells have a micronuclear deficiency. The responsibilityfor the abnormality may be the defect of a specificportion on a specific chromosome in the micronucleus.

Conjugation-Specific RNA as a PossibleTarget Molecule

On the other hand, according to Sugai and Hiwatashi[1974] and Martindale et al. [1985], the synthesis ofmicronucleus-specific RNA shows a peak in stagesII–IV (leptotene to pachytene) but is not detected instage V (diplotene) of the meiotic prophase, althoughthe micronucleus is usually transcriptionally inert inasexual reproduction. However, this does not deny thepossibility for the already synthesized RNA to be foundat stage V. In our experiment, the strongest UV-

Fig. 4. Frequencies of nuclear events in the irradiated cells at 12,15, and 20 h after mixing. At each time, more than 1,000 cells werecounted. Percentage of cells with the normal nuclear development isless than 10%. The sum of the cells with pyknotic micronuclei (Pmic)and the amicronucleates (Amic) always exceeds 80% in frequency.‘‘Others’’ includes cells that developed the abnormal number of anla-gen, transiently reconjugated, and conjugated later than the initialmixing.

UV-INDUCED ABORTIVE CONJUGATION IN TETRAHYMENA 155

sensitivity appeared at stage V, when RNA synthesis inthe crescent micronucleus terminated. The UV-dam-aged molecule in this experiment could be interpretedas the conjugation-specific RNA transcribed from themicronucleus at the meiotic prophase. This interpreta-tion is compatible with that presented by Doerder andShabatura [1980] concerning genomic exclusion. Asmentioned previously, cells in which genomic exclusionoccurs have a deficient micronucleus. Such clones shouldlack a lot of genes in the micronuclear genome, some ofwhich might be conjugation-specific genes. Therefore,we suppose that the lack of such genes results in thegenomic exclusion, probably because of the commondeficiency, as shown in our experiment.

The expression of three conjugation-specific genesand one conjugation-induced gene, which were clonedfrom a cDNA library, was documented [Martindale andBruns, 1983; Martindale et al., 1985]. In that study,conjugation-specific genes showed a maximum level oftheir mRNAs in meiotic prophase. Their peaks inabundance coincided with the stages between III andIV in meiotic prophase, suggesting prophase-specifictranscription. Furthermore, the mRNAs continued toexist even during the stages from metaphase to prezy-gotic division. It has been unclear whether the mRNAsare the same ones as the micronucleus-specific RNAmentioned above, although the temporal correlation ofthe expression of the two RNAs suggests their similar-ity. In any case, these mRNAs can also be possibletarget molecules by UV. In order to ascertain thispossibility, it might be useful to check whether theexpression of the conjugation-specific mRNAs in thepairs with deficient mironucleus is detected.

The results shown in this experiment indicate thatthere is an apparently narrow period of sensitivity toUV and that the window for the execution point of thenuclear abnormalities occurs somewhat later in theconjugation pathway, when meiosis II is completed.How can this time lag between the induction point andthe execution point be interpreted? The transcriptioninhibitor actinomycin D is known to disrupt manyevents of postzygotic development [Ward and Herrick,1996]. The addition of actinomycin D at least 1.5 hbefore each normal event, such as pair separation, oldmacronuclear resorption, and new micronuclear elimi-nation, effectively causes a block. This result suggeststhat this lag is the time from transcription to transla-tion. The time lag approximately coincides with thatobserved in our experiments. This does not deny againthe possibility that the conjugation-specific RNA is apossible target molecule by UV-B.

Comparison With Other Artificially InducedAbnormal Conjugation

This paper showed an example of artificially inducedabortive conjugation. In the UV-irradiated conjugants,an event in the normal conjugation sequence was

apparently blocked, so that further development, includ-ing destruction of the parental macronucleus, was alsoblocked. However, no new micronuclei were retained inthis mating, and the exconjugants became amicronucle-ates and failed to grow. In this respect, this abortiveconjugation is different from genomic exclusion. Rather,in some ways, this may resemble the case in which bothpartners induce genomic exclusion. Other exampleshave been reported in which similar anomalies wereartificially induced by allowing the cells to mate at 40°C[Scholnick and Bruns, 1982] and by mating a wild-typewith the amicronucleate mutant [Kaney, 1985]. Inthese cases, pairs went through normal conjugationuntil the point of nuclear exchange and fertilization,but then anlage development was blocked. These typesof abnormal conjugation, however, are different fromthe abortive conjugation shown in this paper, in thatthe conjugants still degraded their parental macro-nucleus. The related phenomenon has also been pre-sented [Cole and Bruns, 1992]. The authors described anovel mating pathway that combines cytogamy withgenomic exclusion, referred as ‘‘uniparental cytogamy.’’In this mating, inhibition of unilateral transfer of thegametic nucleus by osmotic shock led to degeneration ofthe parental macronucleus in both conjugating part-ners, but to the formation of macronuclear anlagen onlyin one partner. The remaining partners not only de-stroyed their old macronucleus but also failed to de-velop both new macro- and micronuclei. This uniparen-tal cytogamy and the abortive conjugation presented inthe current study are also unlike in some ways.

Finally, Cole et al. [1997] reported a systematicisolation of conjugation mutations. They described fivemutations that affect the earlier stages from meiosis tonuclear selection. cnj4 mutants manifest a phenotypesimilar to that of the UV-induced abortive conjugationin that they abort the subsequent developmental events.However cnj4 mutants seem to have a deficiency inmeiosis II but not in nuclear selection. In our experi-ments, exconjugants with two pyknotic micronucleiwere rarely observed (data not shown). This conditionmight be due to the failure of meiosis II. Judging fromthe low frequency of the occurence of this condition, thecnj4 gene product may not be the main target moleculeby UV. When the exact timing of the expression of thegene is elucidated, the relationship between the cnj4gene product and the UV-sensitive molecule assumed inthis study would be clear.

ACKNOWLEDGMENTSWe express special thanks to T. Sugai for repeated

discussions and a kind supply of his unpublishedresults. We thank K. Hiwatashi and M. Saiki for theircritical reading of the manuscript and helpful sugges-tions. We are also grateful to S. Hoshina for histechnical support on UV irradiation.

156 KOBAYASHI AND ENDOH

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