Maintenance of mating reactivity and capacity of transferring meiotic induction by an activated to a...

THE JOURNAL OF EXPERIMENTAL ZOOLOGY 218~121-126 (1981)

Maintenance of Mating Reactivity and Capacity of Transferring Meiotic Induction by an Activated to a Homotypically United Cell in Blepharisma

GIOVANNI SANTANGELO AND RENZO NOBILI Institute of Zoology and Comparative Anatomy, University of Pisa, Pisa, Italy

ABSTRACT The two complementary mating types of Blepharisma japonicum, I and 11, undergo cell pairing if activated by the complementary gamones. Pairs form in three possible combinations: 1-1, 11-11, 1-11. In the two former unions (homotypic pairs), no nuclear changes occur in the cells. In the third union (heterotypic pairs), a diffusible factor produced during the first 2 hours of cell contact is assumed to induce meiosis and other nuclear changes in the cells. When cells were surgically separated during the first 2 hours after the onset of pairing, the behavior of cells from heterotypic pairs with respect to the occurrence of meiosis was alike in 93% of the cases. Cells of heterotypic pairs, separated from 0.66 to 2.66 hours after mixing complementary mating types, can homotypically reunite with gamone-activated cells of their own mating type. The capacity of separated cells to reunite decreases as the time of heterotypic union increases. In 32 cases out of 80, separated cells whose partner underwent meiosis reunited, and three of them were able to induce macronuclear changes correlated to meiosis in the homotypically united cells.

Conjugation in ciliates brings about a series of developmental changes in paired complementary cells such as meiosis, karyogamy, and nuclear differentiation, as well as morphologi- cal rearrangements. As these cascade pheno- mena proceed, it is not known whether paired cells can maintain the capacity to reunite once separated.

In Blepharisma japonicum, conjugation is preceded by the “preconjugant cell interaction” in which cells of the two complementary types, I and 11, interact with specific gamones (Mi- yake, ’68 Miyake and Beyer, ’73). Type I cells excrete gamone 1, and type I1 cells excrete gamone 2. The two gamones transform the complementary cells so that they can unite. Transformed cells can unite in all three possible combinations of mating types, but only heterotypic pairs (type I-type 11) undergo meiosis and other nuclear changes. Homotypic pairs (type I-type I, type 11-type 11) may remain united for days, without undergoing meiosis or any other nuclear change.

Using a “doublet” strain with two points of attachment for conjugation, Miyake (1975)

was able to obtain chains of cells homotypically united, which behaved as homotypic pairs without nuclear changes. If a cell of the corn- plementary type unites a t one end of the homotypic chain, nuclear changes begin and propa- gate throughout the chain. I t was later found (Miyake et al., ’771, that nuclear activation starts at the site of heterotypic union, propa- gating from cell to cell without skipping any member of the chain. The propagation slows down as it proceeds through the chain, and is slower in longer chains. I t was concluded that a certain substance originating at the site of heterotypic union is the meiosis inducer which can be transferred to the other cells of the chain. We can reasonably assume that the same substance is involved in the meiotic activation of heterotypic pairs.

As far as cell union is concerned, the three types of pairs do not differ from each other at least at the onset of cell pairing. Smaller differences between heterotypic and homotypic pairs are noticed, however, at the cytoplasmic level as cell union proceeds. These differences are related to the size and number of cytoplas-

0022-104X/81/2182-0121$02.0001981 ALAN R. LISS, INC.

122 0. SANTANGELO AND R. NOBILI

mic bridges (larger and more numerous in hetero- vs. homotypic coeval pairs) and in the time of disappearance of the microtubules (much earlier in heterotypic pairs), which are formed at the presumptive area of cell attachment in cells subjected to complementary gamone treatment (Bedini et al., '78; Miyake et al., cited by Miyake, '81).

Blepharisma, which can form both heterotypic and homotypic pairs (one undergoing meiosis and the other not), is therefore an ex- cellent source for studying the meiosis initiat- ing substance. Heterotypic pairs are easily obtained and readily separated experimentally. If a partner of such a pair reunites with a gamone-initiated cell of the same mating type, two things can occur - either the homotypic pair thus formed is or is not induced to meiosis. In the former case the barrier of nuclear activation present in homotypic pairs is overcome.

In this work, we focused on the following two questions: 1) If cells of heterotypic pairs are surgically separated, can they unite homotypically with cells that have never beeh heterotypically united? 2) If this is the case, can the former induce meiosis in the latter?

MATERIALS AND METHODS

Cells Clones R13, R10-3 (mating type 11), and R5-1

(mating type I) of the wild type red strains and clone A5-3 (mating type I) of the albino strain of Blepharisma japonicum var. intermedium were used. All three clones were grown in Hi- watashi's lettuce juice medium (Sonneborn, '70), inoculated with Enterobacter aerogenes and a small ciliate Sapmphilus sp. Cells were concentrated, washed with and suspended in S.M.B. (a salt solution for Blepharisma, Miya- ke and Beyer, '73), and utilized the day after washing. The cultures were grown and the experiments were performed at 24°C f 1 "C.

Gamones Gamone 1 was a sample of Blephormone,

partially purified by Bio-gel P. 150 and C.M. cellulose chromatography and lyophilized, protected by 0.01% bovine serum albumin (Miyake and Beyer, 1974). The solution of gamone 2 was a preparation of synthetic Blepharismone (Tokoroyama et al., 1973) in S.M.B. Stock solutions of both gamones were diluted separately in a 1:99 ratio in S.M.B. before using. Both gamones were a generous gift from Dr. Miyake.

Induction of heterotypic pairs and their isolation

Red cells of either clone R13 or R10-3 and albino cells of clone A5-3 were separately incubated with complementary gamones for about 2 hours in the diluted solution mentioned above. When homotypic pairs started form ing, the beaker in which they were contained was shaken to separate these pairs, and the complementary clones were then mixed.

Heterotypic pairs consisting of a red R13 of R10-3 and an A5-3 albino cell soon formed in the mixture and their number rapidly in- creased. The heterotypic pairs were then isolated and pooled in the cell-free fluid of the mixture. The isolation procedure started 20 minutes after mixing the two types of cells and lasted 20 minutes. Newly formed pairs were so weakly united during the first 10 minutes that they separated on isolation. Such separated pairs were not used. Thus the pairs used in the experiments were mostly those formed within the first 30 minutes after mixing (see Results). The maximum error related to the time of pair formation amounts to 30 minutes, if one takes the mixing as time 0.

Surgical separation and homotypic recognition

The partners of an aliquot of heterotypic pairs were surgically separated every 15 minutes for 2 hours following the isolation procedure. At each time interval, 16 to 58 pairs were cut apart.

Surgical separation was made under dissecting microscope by pressing on the contact area of a pair with a thin, flexible, glass needle. Throughout the entire operation (2 hours) there existed a variety of attachments at the contact area from loose ciliary agglutination (the most easily separated) to closely juxta- posed membranes (maximum distance 20 nm) with minute cytoplasmic bridges. Extreme care was taken not to damage the cells during the separation procedure, and, as the partners were of different colors, all surgically separated cells that showed mixed color at the cut area were discarded. The cutting experiments were performed several times under the closest identical conditions possible, and the data were pooled. The separated red cells of mating type I1 from each pair were individually kept in the medium in which the heterotypic pairs had been isolated. They were used as controls for nuclear changes. Each albino cell from a pair was mixed after the cut with about 200 red

TRANSFEROF MEIOTIC INDUCTION IN BLEPHARZSMA 123

R5-1 cells in 0.5 ml of gamone 2 solution in which they had been previously incubated for about 2 hours (see Gamones). All these mixtures in which an albino cell was present were kept under scrutiny for the appearance of a homotypic pair between the albino and a red cell. All homotypic pairs, as well as the single albino cells that did not pair in the mixtures, were examined for nuclear changes 24-36 hours later.

Nuclear changes Macronuclear changes and meiosis induc-

tion in the surgically separated red cells were ascertained under a dissecting microscope, taking advantage of the differential distribu- tion of the pigment granules around micronuclei (Miyake et al., '77).

Homotypic pairs of red and albino cells and single albino cells were fixed in Sanfelice solution and then stained with Feulgen reagent and light green. Macronuclear changes were then examined in the stained preparations.

RESULTS

Technique The technique used to assay the induction of

meiosis is simple and reliable for red cells but is not applicable to the albino partners. To ascertain whether both partners of a heterotypic pair synchronously undergo activation for meiosis, 54 heterotypic pairs were surgically separated, in groups of nine, every 20 minutes for 2 hours from the onset of conjugation. Each partner cell was kept isolated, and was then examined for meiotic induction: Red partner cells were examined under the dissecting microscope, and albino partner cells were Feulgen- stained and examined under the compound microscope.

In about 93% of the pairs, both partners behaved alike - that is both did or did not undergo meiosis. The 7% discrepancy observed was distributed among the red and the albino cells with either one being activated for meiosis.

This result indicates that the error is not larger than 7% when one assumes the exis- tence of correspondence in meiotic induction between the two cells, albino and red, of a heterotypic pair.

Reconjugation experiments Heterotypic pairs of red and albino cells were

surgically separated every 15 minutes in the time interval of 0.66-2.66 hours after pairing

had started. Each separated albino cell of a pair was immediately placed together with about 200 mating type I red cells in 0.5 ml of medium containing gamone 2. These red cells had been pretreated with gamone 2 and were ready for homotypic cell union. A homotypic pair consisting of an albino and a red cell could then be recognized in the mixture. Once tightly united the homotypic pair was singly isolated in a similar gamone 2-containing medium, followed for meiotic activation of the red partner and later stained for inspection of nuclear changes.

A total of 286 heterotypic pairs were surgically separated, and 80 albino cell partners reconjugated homotypically (Table 1). The red partners of all heterotypic pairs were inspected for meiotic induction, and the data obtained were used to draw the activation curve of Figure 1 in which the percentage (upper curve) of activatedloperated cells (ordinate) is plotted against the time (abscissa) since pairing began. The curve reaches a peak at 1.8 hour after the mixture, which corresponds to the time of meiotic induction in 96% of the operated mating type I1 red cells. The pattern of the activation curve agrees well with similar curves obtained by Miyake et al. (1979) in corresponding cells. In Figure 1 the percentage (lower curve) of mating type I albino cells which homotypically reconjugatedloperated cells is also plotted vs the time of heterotypic union and meiotic activation of red cells. Of the 80 albino cells that homotypically reconjugated, 58 belonged to

TABLE 1. Homotypic reconjugation of cells from operated heterotypic pairs.

Time in minutes Number of cells a b C d

0-15 15-30 30-45 45-60 60-75 75-90

90-105 105-120

Total

36 35 39 58 45 27 30 16

286 -

2 11 0 7 12 2

26 12 4 37 23 12 34 13 7 26 4 3 26 4 3

1 1 13

171 80 32 - - -

a = Heterotypic pairs operated at time 0.66 + t from mixing. b = Activated type I1 red partners of the operated pairs. c = Homotypically reconjugated type I albino partners of the oper-

d = Reconjugated albino cells with their previous red type I1 partner ated pairs.

activated.

124 G. SANTANGELO AND R. NOBILI

Time from the mixture of the two complementary types

Fig. 1. Meiotic activation of heterotypic pairs (upper curve) and homotypic reconjugation of albino separated cells (lower curve).

pairs separated within the first hour and 22 in the second hour of the experimental time. (0.6-1.66; 1.66-2.66 hours) since heterotypic union. The mating type I I red cells induced to undergo meiosis numbered 72 and 99 for the two time intervals, respectively.

The reconjugation curve has higher values at the beginning when the meiotic activation curve shows lower values, and then it drops sharply to almost zero at the leveling off of the meiotic curve.

Among homotypically reconjugated albino cells, 32 out of 80 cells had their previous red partners induced to undergo meiosis. On the basis of the results reported in the first para- graph, one would have expected the corresponding albino cells to be also induced to undergo meiosis. When later analyzed, 27 of them were indeed found to be induced. Out of these 32 homotypically reconjugated pairs, 3 showed meiotic- like macronuclear changes in the red type I partner (Figure 2). The albino partner of these three pairs was separated between 1.25 and 1.75 hours after mixture. The macronuclear changes of these three cells were delayed in comparison with those of their albino partners. As to the micronuclei, no division was ever observed in the red partners while subtler changes could not be detected by the technique used.

DISCUSSION

In Blepharisma japonicum, cell union starts as a ciliary union between the oral ciliature en-

circling the mouth of the cells (Honda and Miyake, '76), followed by oral membrane adhesion and later by the formation of tiny cytoplasmic bridges at the fused membrane level (Bedini et al., 78).

All these modifications, as well as the actual process of cell union, require the presence of complementary gamones which in turn act as promoters of protein synthesis (Miyake and Honda, '76). In heterotypic pairs, both partners are irreversibly committed to meiosis about 1 hour after ciliary union, when cytoplasmic bridges start forming (Miyake et al., '79; Miyake et al. unpublished results). I t was not known whether heterotypic membrane fusion and meiotic induction, which later ensues, inhibit the capacity of cells to reunite again when experimentally separated.

In Chlamydomonas the flagellar tip agglutination (Goodenough and Weiss, '75), which is mating type-specific, comes apart and never resumes once cell body membrane fusion has occurred.

The results reported here clearly indicate that the cells, separated from heterotypic union, are still capable of uniting homotypically (80 out of 286 cells) if mixed with cells of the same mating type, previously activated by gamone for conjugation. The longer the cells have been heterotypically united, the fewer are able to reunite, thus showing a strong correlation with the progression of meiotic induction. This correlation is quite apparent in the results of Figure 1 upper and lower curves. However,

TRANSFEROF MEIOTIC INDUCTION INBLEPHARISMA 125

Fig. 2. Homotypic pair in which the albino type I cell (at the left) shows a quite contracted macronucleus and the red type I cell (at the right) shows a coiled macronucleus. Feulgen staining. X 250.

27 out of 80 separated cells, which underwent cell union again, proved to be irreversibly induced to meiosis. It can therefore be reasonably assumed that the modifications and the state of the surface membrane required for cell union (whatever they are; cf. Miyake, '78) per- sist even in cells already committed to meiosis. As the meiotic process further progresses, the ability to pair, at least homotypically, is lost; however, whether these cell unions had passed through the normal steps of ciliary and membrane adhesion is not known. The cut itself cannot be responsible for the reunion of the cells, as the reunion drops to zero in separated cells after 2.6 hours of heterotypic pairing.

Bedini et al. ('78) have recently provided evidence that an extensive breakdown of microtubules and other cytoplasmic structures occurs at the heterotypic united area of pairs as old as about 3 hours and already committed to meiosis. Such a breakdown does not occur, or it occurs much later at the united area of homotypic pairs. Other evidence shows that cells of

homotypically united pairs can unite once again homotypically when separated after many hours of pairwise union (personal observation).

Whether the failure of separated heterotypic paired cells over 2 hours old to reunite homotypically is associated in our case with that breakdown and/or with the inability to rebuild such structures, although a very suggestive hypothesis, remains yet to be determined.

The second question posed in the introduc- tion was to ascertain if cells induced to meiosis were capable of transferring their state of induction to cells with which they reunited homotypically. In this connection we have to consider the 32 albino cells, presumably committed to meiosis, and especially the 27 cells that indeed passed through meiosis and had reconjugated homotypically. In three cases of reconjugation, macronuclear changes very similar to those associated with meiotic activation were observed in the red type I partners. In spite of the low number of activated red cells, this result indicates that meiotic-like nuclear changes can be induced in homotypic pairs, provided that one partner is activated prior to conjugating again homotypically. This fact is consistent with the findings of Miyake et al. ('77) with respect to the propagation of meiosis and other nuclear changes between cells of homotypic chains to which a complementary cell is united at one end.

The failure of induction of nuclear changes in more homotypically paired cells than the three observed is not a contradiction of what was stated above. In the chain system, no physical barrier to meiosis propagation exists between the inducer and the inducible; that is, the next cell of the chain not yet activated. In the present two-cell system the would-be inducer, if activated at the cutting time, unites with the inducible cell at least 1 hour after it has been activated to meiosis. Moreover, the new homotypic pair should reach the stage of a very inti- mate union with cytoplasmic bridges compar- able to the homotypic union between cells of a chain (Bedini et al., unpublished result) about 5 hours after the reconjugation began. If the activated partner stops producing the trans- ferrable factor for meiosis as soon as it is cut away from the heterotypic union, or even as soon as the partner cell is activated, the factor must remain active for 6 hours to induce nuclear changes in the partner. This value is similar to that found by Miyake et al. ('77). In our case it corresponds to the shortest time observed when a separated cell immediately reunited homotypically. The majority of separated cells took a longer time to reunite homo-

126 G. SANTANGELO

typically and it is not surprising. therefore, to find that macronuclear changes appeared in the red type I cell of only three pairs. This low efficiency could also be explained by assuming that the substance was unstable. If the substance breaks down after heterotypic separation, the longer the homotypic pair takes to reach the right stage, as mentioned previously, the lower the amount of the substance that passes to the partner. The amount of the factor that passes to the partner may also be relevant as to how meiosis and other nuclear changes progress in the inducible member. The stage of the macronuclear changes reached by the three cells, considered to be activated, lagged behind that expected after 24 hours of cell union (Figure 2). If this lag is due to the low amount of the factor passed into the cell, any delay in reconjugation would be expected to lower the amount still further, so that no sign of activation could be expected to become apparent at the macronuclear level. Nor could our method detect micronuclear changes that do not com- plete the meiotic process. For these reasons, partially activated cells may have escaped our analysis.

AND R. NOBILI

ACKNOWLEDGMENTS

Our most sincere thanks are due Dr. A. Miyake for interesting discussions, critical revision on the manuscript, and for providing gamones and new strains of Blepharisma

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