Conjugation in Tetrahymena thermophila

Copyright @ 1982 by Academic Press. Inc. All rights of reproduction in any form reserved 0014-4827/82/070227-10$02.00/O

Experimental Cell Research 140 (1982) 227-236

CONJUGATION

IN TETRAHYMENA

THERMOPHILA

A Temporal Analysis of Cytological DUANE W. MARTINDALE,L

Stages

* C. DAVID ALLIS2 ** and PETER J. BRUNS

‘Section of Genetics and Development, Cornell University, Ithaca, NY 14853and =Department of Biology, University of Rochester, Rochester, NY 14627, USA

SUMMARY The time course and synchrony of the stages of conjugation in Tetrahymena thermophila as defined by cytologically observable changes in the morphology and position of the nuclei were established. The time required for 50% of the pairs to enter or pass a particular stage, as well as the duration of each stage were determined. The relative synchrony of the pairs as they went through conjugation was followed by correlating the maximum percentage of the population found in a stage with the duration of that stage. The degree of synchrony between the pairs was found to be high under the conditions of this study, with very little decrease in synchrony seen during the initial 9 h of conjugation. Although some variability in the degree of synchrony was seen between different matings, there was little change detected in the duration of each c tological stage. Prolonged starvation of the cells prior to their mating resulted in a gradual loss ofysynchrony.

Conjugation in the unicellular protozoan jugation [ 1,2, 4, 10-121, but the correspondTetrahymena thermophila is an orderly de- ing cytological studies have been mainly velopmental process during which cell pair- descriptive and give only estimates of the ing, meiosis, genetic exchange and forma- time course of conjugation [13-161. Since tion of new somatic (macro) and germinal many of the biological processes that occur (micro) nuclei occur [ 131.For a mature cell during conjugation in Tetrahymena are beto begin conjugation it must be Ftarved for ginning to be analysed by biochemical ‘and a period of time (‘initiation’j [ 1, 23. Once molecular techniques, a quantitative analyinitiated it must physically interact with sis of the cytological stages of conjugation initiated mature cells of another mating type was initiated. The time of their onset, dura(‘co-stimulation’) [3, 41. Once cell pairing tion and the degree of their synchrony are has occurred, the processes necessary for reported here. sexual reproduction occur and are followed by nuclear differentiation. The ease with MATERIALS AND METHODS which Tetrahymena can be grown and manipulated genetically and biochemically Strains (e.g. [5, 61) has made it attractive for in- The strains of T. thermophila used were CU355 and CU356; both are genetically marked strains derived vestigating many of the events occurring in from inbred strain B. conjugation, such as cellular recognition To whom offnrint reauests should be addressed: and interaction [4], rDNA amplification [7, *Section of Gen&ics and’Development, 203 Bradfield Hall, Cornell University, Ithaca, NY 14853, USA. 81, and nuclear differentiation [9]. ** Current address: Department of Biochemistry, Several studies have reported the kinetics Baylor College of Medicine, Houston, TX 77030, of the pre-pairing and pairing events of con- USA. Exp Cell Res 140 (1982)

228

Martindale, Allis and Bruns .

loom---,

Conjugation Cultures were starved for at least 24 h. By this time cells have recovered from the initial shock of the wash into starvation medium and the background synthesis of protein and RNA has reached a minimum [18]. To begin conjugation, equal numbers of cells from two starved cultures of different mating types were gently mixed at 30°C in an Erlenmeyer flask ten times the volume of the mating mixture. The concentration of the mating mixture was 1.5-3x 105cells/ml.

Cytology 1). 1 Oo

2

3

4

5 6 7 hours since

8 9 mixing

10

11

12

13

14

Fig. I. Pairing kinetics. The average of three experiments with SD indicated (A) is superimposed over the results from expt 1 (O), in which the most time points were taken. The conditions in the three experiments were identical, except for the duration of starvation (24 h in expt I, 25 h in expt 2 and 40 h in expt 3).

Culture conditions The cells were grown, starved and mated at 30°C. The medium used for growing the cells was that described by Leick, Engberg & Emmersen [17], but diluted to one-third of the original concentration. Final concentrations were 0.25% proteose peptone, 0.25% yeast extract, 0.5% glucose, 0.3 mM magnesium sulfate, 17 PM calcium chloride and 33 FM ferric chloride or ferric citrate. Doubling time in this medium for these strains was 2.5 h, with exponential growth continuing to a cell concentration of over 5x 105cells/ml. To initiate starvation, cultures were collected and washed once with 10 mm Tris-HCI, pH 7.4, by centrifugation at 100 g for 2 min in a clinical centrifuge at room temperature. Cells were resuspended at a concentration of approx. 2x105 cells/ ml in an Erlenmeyer flask in a volume between onetenth and one-fifth the flask volume. It was not necessary to agitate cultures during starvation.

At each time point, 200 ~1 samples of conjugating cultures were stained using the Giemsa method described by Bruns & Brussard [a].

RESULTS Kinetics of pairing Data for the kinetics of cell pairing from three separate experiments are plotted in fig. 1. The average results with standard deviation are shown superimposed over the results from expt 1. We were consistently able to obtain over 90% pairing. These results are in agreement with those in previously published reports, even though different strains and starvation media were used[l, 11, 191. A small burst of cell division coincident with the onset of pairing was seen. This phenomenon has been observed by others [3, 191 and appears to be necessary for advancing some cells to macronuclear Gl. Fig. 2. A diagrammatic illustration of the postmeiotic events of conjugation. a, Meiosis produces four haploid nuclei; b, mitosis of one of the haploid products produces pronuclei; c, exchange of haploid pronuclei and destruction of the remaining haploid nuclei; d, fertilization; e, two mitotic divisions of the zygote nucleus;f, visible development of new macronuclei (macronuclear ‘anlagen’) begins; g , continued development of macronuclear amagen and destruction of the old macronucleus; /I, pair separation; i, cellular division yields four caryonides.

Exp Cell Res 140 (I 982)

Analysis of Tetrahymena

Fig. 3. The cytological stages of conjugation. a, Micronucleus no longer closely associated with macronucleus; b, meiotic prophase stage I (see text); c, stage II; d, stage III; e, stage IV (full crescent); f, stage V; g, stage VI; h, meiotic metaphase; i, first prezygotic dtvision; j, k, second prezygotic division; I, m, third prezygotic division; the putative migratory

conjugation

229

(m) and stationary (s) pronuclei are labeled; n, o, first postzygotic division; p, q. second postzygotic division; r, macronuclear development I; S, t, macronuclear development II. The variable size of the paired cells is accented by the different degree to which the cells were flattened in different slide preparations. Bar, (t) 50 pm.

the stage at which conjugation may occur

3b-g). During this stage, the ten (n=5) bivalent chromosomes apparently pair and POI. join in a unique linear configuration called Cytological events the crescent (fig. 3e) [14, 161. This is followed by three prezygotic nuclear divisions Cytologically, conjugation in T. thermophila consists of meiotic prophase, three consisting of two meiotic divisions (fig. prezygotic nuclear divisions, two post- 3tk) followed by one mitotic division (fig. zygotic nuclear division, and macronuclear 31). The meiotic divisions reduce the didevelopment [ 131.Fig. 2 diagrams the major ploid chromosome number to haploid; only postmeiotic events of conjugation. Fig. 3 one of the resulting four haploid nuclei is presents micrographs which illustrate the retained. The remaining nuclei (‘relic’ nucytological stages in detail. Meiotic pro- clei) begin to disintegrate just prior to the phase occurs soon after cell pairing (fig. third prezygotic nuclear division and a few Exp Cell Res 140 (1982)

230

Martindule. Allis and Bruns

jugation, cytological stages were defined in the following manner. The meiotic prophase was divided into the six stages defined by Sugai & Hiwatashi [14]. This classification is based upon the stage of elongation or contraction of the micronucleus; stage I (fig. 36) begins when the micronucleus starts to elongate, stage IV (fig. 3e) includes the maximum elongation of crescent stage, and stage VI (fig. 3g) ends when the bivalent chromosomes separate. Metaphase (fig. 3h) includes all stages from the end of prophase stage VI to anaphase I. A particular prezygotic or postzygotic nuclear division was defined as beginning when an anaphase configuration was seen. Thus, the third prezygotic division encompasses the events beginning with 0 1 2 3 4 5 k--e-e9 hours since mixing the anaphase configuration of the nucleus Fig. 4. Pairs in a particular stage vs time since mixing. that will form the migratory and stationary Each division on the ordinate represents a difference of 10%. The baseline for each stage is placed 10% nuclei (fig. 31) and ending just before the above the baseline of the previous stage. Small roman anaphase configuration of the synkaryon in numerals designate the six stages of meiotic prophase. the first postzygotic division (fig. 3n). The Between 101 and 178 pairs were examined every halfhour. description of the pre- and postzygotic nuclear divisions by Ray [ 131was very useful may remain to the beginning of the second in deciding which stage to assign to a parpostzygotic nuclear division. The mitotic ticular conjugating pair. When the developdivision produces a migratory pronucleus mental stages in the two members of a pair (m) and a stationary pronucleus (s) (fig. were slightly different from each other, the 3m) from the retained haploid nucleus in pair was designated as being in the later each conjugant. The two members of each stage. Macronuclear development I (fig. pair exchange migratory pronuclei (fig. 2f, 3r) was defined as beginning after the 3m), and the subsequent fusions of migra- second postzygotic division when the two tory and stationary pronuclei yield diploid anterior nuclei (macronuclear anlagen) bezygote nuclei (synkaryons). In each con- came visibly larger than the two posterior jugant, two postzygotic divisions of the syn- nuclei. Macronuclear development II (figs karyon follow (fig. 3n-q), yielding four nu- 2g, 3s) was defined as beginning when the clei. The posterior two develop into micro- old macronucleus moved to the posterior of nuclei, while the anterior two (macronu- the micronuclei while the micronuclei clear ‘anlagen’) develop into macronuclei moved anteriorly to lie between the macro(fig. 3r-t). The old macronucleus begins to nuclear anlagen. degenerate soon after the new macronuFig. 4 graphically illustrates the results of clear anlagen begin development. an experiment in which a mating mixture To quanfitate the various events of con- was examined cytologically every half-hour Exp Cell Res 140 f/982)

Analysis of Tetrahymena conjugation

231

Fig. 5. Consensus curve obtained by

superimposing data from each curve in fig. 3. The ordinate gives the relative fraction of pairs in a certain stage, with the maximum percentage equalling one. The negative numbers on the abscissa indicate the time before the maximum occurs. Solid line, normal curve; dotted line, best fit. The data are from expt 1. -1

-.5

0

1

.5 HOURS

FROM

1.5 2 MAXIMUM

2.5

3

after pairing began. At each time point. the layed, causing a trailing-off of the conpercentage of the pairs in each cytological sensus curve. stage was determined. With this type of Data from expt 1 were replotted as pergraph, both the maximum percentage of the centage pairs in or past a specific stage pairs that would be expected in a particular vs time after mixing the prestarved parents stage, and the time when this maximum oc- (fig. 6). Fig. 6 allows a determination of the curs can be determined. The curves seen in time when 50% of the population has fig. 4 can be superimposed upon one an- entered a particular stage (50% entry time). other if the maximum in each stage is set to Measuring the time between any two stages 1.0. Fig. 5 shows the consensus curve that allows a determination of the duration of is obtained; a normal curve has been super- the earlier stage. Because of the slight lag in imposed on this curve. We conclude that a small portion of the population, the 50% two-thirds of the pairs (determined by entry time for each stage is slightly later measuring the area below the normal and than the time of its maximum distribution experimental curves) entered each stage (% max). with a normal distribution, whereas oneIn addition to expt 1, the two matings third of the population was somewhat de- that were used for the kinetic analysis of

Fig. 6. Pairs in or past a particular stage

vs time since mixing. The roman numerals represent the six stages of meiotic prophase (see text). The data were obtained from expt 1.

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Table 1. Quantitative analysis of conjugation stages Stage Cell pairing Meiotic prophase I II III IV V VI Diplotenelmetaphase Prezygotic divisions 1st 2nd 3rd Postzygotic divisions 1st 2nd Macronuclear development I II Cells come apart Total duration Cell pairing Meiotic prophase Prezygotic divisions Postzygotic divisions

Maximum in stage (%)

Time 50% entered (min)

Stage duration (min)

76k 7

606f 16

54+23” 40f17 29+11 412 4 18t 7 14+ 1 18f 4

97228 139f22 176+20 2024 17 245f26 257f27 267f28

43+ 7 37+12 26+ 4 43+ 10 13f 2 10f 2 14+ 3

23flO 42+ 9 36+ 9

281+30 299f33 333f34

18+ 4 34f 3 39k 2

37f 8 26+ 4

372535 404+35

32f 1 25f 7

29f 10

429f41 46Of37 684t24

31f 5

606+16 185f 7 91f 6 57+ 7

’ The data shown are the mean values of three independent experiments plus or minus their SD.

pairing (fig. 1) were also examined cytologically. The 50% entry times as well as the duration of each stage were determined for all three experiments. These data are tabulated in table 1 as mean values plus or minus the standard deviation, and are graphically illustrated in fig. 7. The time at which 50% of the pairs entered comugation varied significantly between experiments, probably reflecting differences in synchrony between the experiments (see below). In marked contrast, the actual length of the various cytological stages is very nearly the same for all experiments. Thus there was variation between the experiments for when the process began, but, once started, the three populations proceeded through the developmental sequence with reproducible kinetics. The Exp Cell Res 140 (1982)

variability in the stage durations among the first four stages of meiotic prophase may be due to the difficulty in distinguishing when one of these stages stops and the next one starts. It should be noted, however, that the total length of meiotic prophase shows the same low variability as the total lengths of the prezygotic and the postzygotic divisions. In a small portion of the pairs (2-5%) the synkaryon did not appear to undergo postzygotic divisions. If these pairs come apart and survive without the postzygotic nuclear divisions the genetic consequence would be that a portion of the population would have made a new micronucleus without making a new macronucleus. Using genetic techniques, S. Scholnick (personal communication) observed what may be the

Analysis of Tetrahymena

conjugation

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Fig. 7. The timing of cytological stages in three separate experiments. Each stage is shown to begin at the time by which 50% of the pairs have entered the indicated stages. The letter M under meiotic prophase represents the first meiotic metaphase.

same phenomenon. He found that threequarters of the cells that do not express a new phenotype after pairing have a new micronucleus. These results indicate that in a ‘normal’ cross, a small percentage of the pairs make new micronuclei but abort macronuclear development. This process may be similar to round one of genomic exclusion [21] in which the majority of pairs in a cross of a normal strain with a defective (‘star’) strain retain their old macronucleus while making a new self-fertilized and thus homozygous micronucleus. An estimate of synchrony was obtained by estimating the maximum percentage of the pairs. found in each particular stage (% max) and comparing this value with the duration of that stage. The more synchronous a population, the higher the % max for a stage of a given duration. The data from the three matings are plotted in fig. 8 as % max versus stage duration. A linear relationship is seen, with the conjugation in expt 1 being the most synchronous of the three. Since synchrony is directly proportional to the ratio of % max to stage dura-

tion, one may ask whether synchrony decreases with time during conjugation. This is accomplished by plotting this ratio for each stage vs the time that the stage occurred (fig. 9). The data points are somewhat scattered because the ratio of two estimations, each containing error, are being

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8. 'Ihe maxtmum percentage 01 cells that were found in any particular stage vs the duration of that stage; a measure of synchrony between experiments. Linear regression was used to draw lines through the data from experiments one (O), two (W), three (A), and the combined data of all three experiments (ave).

Fig.

ExpCeNRes

140 (1982)

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Martindale,

Allis and Bruns .. .

2.0

o.s_ 0

400

loo MINUTES

SINCE

500

MIXING

9. The change in synchrony during conjugation. The average time by which 50% of the pairs had entered a stage was determined from the three experiments and this value was used when plotting the ratio of that stage. Linear regression was used to draw lines through the data from experiments one (O), two (M), and three (A).

Fig.

plotted. A slight decrease in synchrony with time after mixing parental cells was seen in all three experiments, with the decrease being most pronounced in expt 1. The effect that prolonged starvation has upon the synchrony of conjugation is seen in fig. 10. The percentage of the pairs in the different stages of conjugation was determined 6 h after starved parental cells were mixed. The parental cells were starved separately for the same length of time; either 25 h, 40 h, 7 days, or 13 days. Synchrony was drastically reduced after the cultures were starved for 2-7 days before they were mixed. DISCUSSION This paper presents a quantitative analysis of the cytological stages of conjugation in Tetrahymena. The time that the stages occur, the length of the stages, and the maximum percentage of cells found in each stage were determined. The synchrony between and within matings was also examined. Exp Cell Rrs 140 (1982)

Our analysis was complicated by the fact that a small fraction of the cells proceed through conjugation later than the majority of the pairs. Since this ‘shoulder’ on the curve in fig. 5 was not observed to change significantly with progressive stages, it is probable that it represents a population that begins the events of conjugation later, but having begun, proceeds through the stages at a similar rate to that of the main population. Since there is evidence that T. thermophifa must be in macronuclear Gl to conjugate [19, 20, 221, and a significant fraction of a culture of Tetrahymena may be in G2 after 24 h starvation [23], the delayed population may contain those cells which were not in Gl at the time starved parental cells were mixed. The cells not in Gl at the time of mixing may be responsible for the small amount of cell division seen in this and other studies [3, 191 soon after the different mating types come in contact. The curve drawn by Orias and coworkers [25] illustrating the time during conjugation when an osmotic shock prevents nuclear exchange also shows a delay in some of the population. Further, the timing of their curve closely corresponds to the curve drawn for the third prezygotic division (fig. 4) during which nuclear exchange occurs. When the conjugation data were plotted on probability paper as the percentage of pairs in or beyond a certain stage vs time [24], a large deviation from the expected straight line was seen above 60% because of the subpopulation of pairs delayed in entering conjugation, and there were not enough points below 60% for each stage to draw an accurate line. Therefore, the relative synchrony between and during experiments was monitored by estimating the percentage of pairs in a stage at the time of its maximum distribution (% max) and cor-

Analysis of Tetrahymena conjugation

235

IO. The effect of prolonged starvation upon the synchrony of conjugation. Cytological stages of conjugation on ab scissa: a, micronucleus in a pocket of macronucleus; b, micronucleus out of macronuclear pocket, c-h, meiotic prophase stage I (cl, II (4, III (e), IV W, V (gh and VI (h); i, diplotene to metaphase I, j, first prezygotic nuclear division; k, second prezygotic nuclear division; I, third prezygotic nuclear division; m, first postzygotic nuclear division; n, second postzygotic nuclear division. Cell cultures of different mating types had been starved in 10 mM Tris-HCl (pH 7.4) for either 25 h (fig. lOA); 40 h (fig. 10B); 7 days (tig. 1OC); or 13days (fig. 1OD). At least 119pairs were examined for each mating. Fig.

D. 13 dap i

t

STAGE

OF CONJUGATION

relating this value with the duration of the stage. The higher the synchrony of a population, the higher the % max for a stage of a given duration. A completely synchronous population would have a % max of 100% no matter what the duration of the stage was. In this study, the synchrony of conjugation was seen to vary between matings (fig. 8), as did the time by which 50% of the pairs had entered the different stages of conjugation. It is not clear what factors were responsible for this variability. Efforts were made to keep all of the conditions the same (temperature, cell concentration, culture volume, flask volume, state of cells when starvation was initiated, etc.). The only obvious parameter that varied was the duration of starvation, but the degree of

synchrony of the cultures starved 25 h was more similar to that of the culture starved for 40 h than that of the culture starved for 24 h. Synchrony of conjugation was lost when the cultures were starved for more prolonged periods (fig. 10. The synchrony of conjugating cells appeared to decrease only slightly during mating (fig. 9). This work was supported by grants from NIH (Gh427871) and NSF (PCM77-07056) to. P. J.B. D. W. M. was the recipient of a NATO Science Fellowship. Part of this work was carried out by D. W. M. in the laboratory of R. E. Pearlman at York University in Toronto. We thank Dr Pearlman for his hospitality and Drs Pearlman and H. M. Martindale for advice and critical comments.

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2. Wellnitz, W R & Bruns, P J, Exp cell res 119(1979) 175. 3. Bruns, PJ & Palestine, R F, Dev bio142 (1975) 75. 4. Finley, M J & Bruns, P J, Dev biol 79 (1980) 81. 5. Orias, E & Bruns, P J, Methods in cell biol 11 (1976) 248. 6. Bruns, P J & Brussard, T B, Science 213 (1981) 549. 7. Pearlman, R E, Andersson, P, Engberg, J & Nilsson. J R. Exo cell res 123(1979) 147. 8. Yao, MIC, Cell 24 (1981) 765. . ’ 9. Gorovsky, M A, Ann rev genet 14 (1980) 203. 10. McCoy, J W, J exp zoo1 180(1972) 271. 11. Allewell, N M, Gles, J & Wolfe, J, Exp cell res 97 (1976) 394. 12. Allewell, N M &Wolfe, J, Exp cell res 109 (1977) 15. 13. Ray, C Jr, J protozool 3 (1956) 88. 14. Sugai, T & Hiwatashi, K, J protozool 21 (1974) 542. 15. Doerder, F P & DeBault, L E, J cell sci 17 (1975) 471.

Exp Cell Res 140 (1982)

16. Wolfe, J, Hunter, B & Adair, W S, Chromosoma 55 (1976) 289. 17. Leick, V, Engberg, J & Emmersen, J, Eur j biochem 13 (1970) 238. 18. Martindale, D W. Unpublished results. 19. Wolfe, J, Dev biol 35 (1973) 221. 20. - Ibid 54 (1976) 116. 21. Allen, S L, Genetics 5.5(1967) 797. 22. Wolfe, J, Exp cell res 87 (1974) 39. 23. Salamone. M F & Pearlman. R E. Exp_ cell res 110 (1977) 32i. 24. Nordby, @ & Nordby, G, Exp cell res 103 (1976) 31. 25. Orias, E, Hamilton, E P & Flacks, M, Science 203 (1979) 660.

Keceived December 4, 1981 Revised version received January 8, 1982 Accepted January 12, 1982

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