ABSTRACT
The expression of sexual activity in Paramecium caudatum is repressed for about 50 fissions after conjugation. The ability of the macronucleus to affect the expression of sexual activity according to the age of clonal development was investigated using the macronuclear fusionreorganization method. When a mature macronucleus was transplanted into an immature cell and fused with a macronucleus in the immature cell, the clones derived from the recipient showed sexual immaturity. In a reverse experiment, an immature macronucleus was transplanted into a mature cell, and the clones also showed sexual immaturity. The ability of the macronucleus to transform mature cells to immature cells was clonal agedependent. The characteristics of the immaturemature hybrid macronucleus indicate that the immature macronucleus is dominant over the mature macronucleus with respect to the ability to express sexual activity. On the other hand, in cells of the early immaturity period, the micronucleus, known as the germ nucleus, shows the ability to undergo meiosis and eventually to produce progeny under control of the mature macronucleus. The expression of sexual activity is thought to be governed by the clonal age of the macronucleus and not by the clonal age of the micronucleus or cytoplasm. The macronucleus seems to determine the ability to express sexual activity by counting postconjugation divisions and keeping track of the age of the clone.
INTRODUCTION
Sexual maturation during clonal development in Paramecium is based on an unique system of intrinsic temporal programming related to clonal aging (Sonneborn, 1957; Nanney, 1980). As in many metazoa, the ciliated protozoan Paramecium shows a change from sexual immaturity to maturity over the course of its life cycles (Sonneborn, 1957; Miwa and Hiwatashi, 1970; Bleyman, 1971). The length of the immaturity period in this organism is genetically controlled and measured in terms of the number of cell divisions after conjugation (Miwa and Hiwatashi, 1970; Myohara and Hiwatashi, 1978). In Paramecium caudatum, sexual activity is repressed for 50-60 fissions after conjugation, as determined by a mating reactivity test using matingreactive cells of a complementary mating type (Miwa and Hiwatashi, 1970). Biochemical studies have shown that sexual activity in Paramecium is associated with the ciliary membrane proteins, called mating substances, that recognize complementary mating types and promote cell contact at the beginning of conjugation (Takahashi et al., 1974).
Our previous studies demonstrated that the cytoplasmic factor, immaturin, which is a heatlabile soluble protein with a molecular mass of about 10 kDa, represses sexual activity when injected into sexually mature cells until it is lost or diluted over the course of cell division (Miwa et al., 1975; Haga and Hiwatashi, 1981). In addition, the microinjection of immature cytoplasm indicates that immaturin activity reveals a peak and then declines during the course of the immaturity period (Miwa, 1984).
An alternative approach to the study of intrinsic temporal programming in sexual maturation is nuclear transplantation. Paramecium has two kinds of nuclei: a micronucleus and macronucleus. The macronucleus is comparable to the somatic nucleus in metazoan cells and the micronucleus is equivalent to a germ nucleus. Although both micro- and macronuclei replicate in each cell division, only the macronucleus is transcriptionally active, and therefore the phenotype of the cell is thus determined by this nucleus. In clonal development, the age of the nucleus is functionally equivalent to the number of nuclear divisions after conjugation. However, it is unknown whether both micro- and macronuclei require the same number of vegetative fissions in the immaturity period before the cell becomes sexually mature.
The present study examined the role of the micro- and macronucleus in the intrinsic temporal programming related to the expression of sexual activity. Our findings indicate that the macronucleus counts postconjugation divisions and keeps track of the age of the clone in sexual maturation, and that the micronucleus is not involved in the programming of sexual maturation.
MATERIALS AND METHODS
Culture conditions
Three strains of Paramecium caudatum, syngen 3, were used: C103, 16B1104, and 16A1015. The phenotypes of these stocks are shown in Table 1. The culture medium was 1.25% (w/v) fresh lettuce juice diluted with K-DS (Dryl’s solution modified by the substitution of KH2PO4 for NaH2PO4), pH 7.0 (Dryl, 1959), and inoculated with Klebsiella pneumoniae one day before use (Hiwatashi, 1968). Cultures were maintained at 25°C.
Macronuclear transplantation and assay of macronuclear fusionreorganization
Macronuclear transplantation was performed after the methods of Koizumi (1974), modified by Haga et al. (1984). The whole macronucleus (approximately 40 pl) was transplanted. Recipient cells were maintained at 25°C for about 12 hours in a cell-free culture medium that was prepared by filtering the culture medium through a 0.22 μm millipore filter on the first day of the stationary phase. The recipients were then transfered into fresh culture medium. Two fissions after the transplantation, 4 sister cells were isolated and cloned separately in test tubes.
Nuclear fusion was tested with a DNA-specific fluorescent dye (H33258). After macronuclear transplantation, recipients were incubated in cell-free culture medium with 5 μM H33258, and observed under a fluorescence microscope at 2, 6, and 12 hours after the transplantation.
RESULTS
Principles of experimental methods
The macronuclear fusionreorganization method is shown schematically in Fig. 1. Macronuclear fusion-reorganization, which corresponds to the process occurring after fusion of a ‘foreign’ (injected) macronucleus with the ‘resident’ macronucleus was examined by testing for swimming behavior and mating-type. A complementation test of cnr genes was performed at about 4 and 15 fissions after transplantation. Swimming behavior was tested in K-DS containing 20 mM KCl. In this test, wild-type cells swim backward for 40-60 seconds while cnrA and cnrB cells do not. The mating-type of each subclone was tested in the test tube culture by mixing the cells of complementary mating-type cells. The behavioral mutants, cnrA and cnrB, were used as testers of mating reactivity (Takahashi, 1979). A quantitative assay of mating reactivity was performed by using the glass capillary method (Haga and Hiwatashi, 1981). In this test, the mating reactivity of each clone was expressed as the percentage of matingreactive cells. The method used to produce heterokaryons is shown in Fig.2. Heterokaryons were produced by the conjugation of cnrA cells and cnrB cells. In normal conjugation, the clones derived from exconjugant cells contain cells whose micro- and macronucleus are newly produced from a synkaryon (NR: nuclear reorganization). However, in the case of P. caudatum, the macronucleus is sometimes regenerated from a fragment of the parental macronucleus (MR: macronuclear regeneration)(Mikami and Hiwatashi, 1975). In these cells, the micronucleus is produced from the synkaryon, but the macronucleus is produced from the fragment of the parental macronucleus, resulting in a cell with a new micronucleus and an old macronucleus. When MR occurs in one of the daughter cells, two types of cells are produced in the clones: one is NR and the other is MR. Since the parental macronucleus makes cells CNR, MR produces CNR cells. This CNR must be a heterokaryon, i.e. its macronucleus is old and its micronucleus is young. The genetic characteristic of the micronucleus in the CNR cell is wild-type while that of the macronucleus is either cnrA or cnrB. When these heterokaryons are mixed with the cells of complementary mating types, they conjugate because the macronucleus is derived from that of a mature cell. In the MR cells, the cellular phenotype controlled by the macronucleus is CNR and sexually mature, while the micronucleus is wild type in behavioral genotype and in the same developmental stage as the immature cell. If the micronucleus of the heterokaryon has the ability to produce true progeny, wild-type cells would be obtained by intraclonal conjugation (selfing conjugation) of the heterokaryon.
Macronuclear fusionreorganization method. The macronucleus from a cnrB cell is injected into a cnrA cell. Recipients were incubated in cell-free culture medium at 25°C overnight. During incubation, the injected macronucleus fused with the macronucleus of the recipient. After nuclear fusion, cells were incubated in fresh culture medium. Two fissions after macronuclear fusionreorganization, cells were isolated and subclones were grown.
Macronuclear fusionreorganization method. The macronucleus from a cnrB cell is injected into a cnrA cell. Recipients were incubated in cell-free culture medium at 25°C overnight. During incubation, the injected macronucleus fused with the macronucleus of the recipient. After nuclear fusion, cells were incubated in fresh culture medium. Two fissions after macronuclear fusionreorganization, cells were isolated and subclones were grown.
Production of a heterokaryon by macronuclear regeneration (MR), and progeny of the heterokaryon. Mating pairs of cnrA and cnrB were isolated in K-DS and incubated at 25°C for 48 hours. Then, exconjugants were incubated in fresh culture medium and grown. About 3 fissions after conjugation, CNR cells were isolated according to behavioral testing and cloned. About 14-15 fissions after cloning, CNR cells were mixed with wild-type cells of complementary mating-type and intraclonal matingpairs of CNR cells were isolated by behavioral testing. Several fissions after conjugation, the behavioral phenotype of the progeny was determined using the test solution described in Materials and Methods.
Production of a heterokaryon by macronuclear regeneration (MR), and progeny of the heterokaryon. Mating pairs of cnrA and cnrB were isolated in K-DS and incubated at 25°C for 48 hours. Then, exconjugants were incubated in fresh culture medium and grown. About 3 fissions after conjugation, CNR cells were isolated according to behavioral testing and cloned. About 14-15 fissions after cloning, CNR cells were mixed with wild-type cells of complementary mating-type and intraclonal matingpairs of CNR cells were isolated by behavioral testing. Several fissions after conjugation, the behavioral phenotype of the progeny was determined using the test solution described in Materials and Methods.
Characterization of the macronucleus reorganized after nuclear fusion upon transplantation
In order to determine whether the two macronuclei brought together by transplantation were completely mixed and reorganized, two behavioral mutants, cnrA and cnrB, were used in which three genetic markers, cnrA, cnrB and Mt
available. Macronuclei of 16B1104 (Mt/Mt, cnrB/cnrB) were transplanted into 16A1015 (mt/mt, cnrA/cnrA) and the recipients were incubated with a DNA-specific fluorescent dye and then periodically observed under a fluorescence microscope. At 2, 4, and 6 hours after transplantation, neither macronuclear fusion nor behavioral phenotypic changes were observed. About 12 hours after transplantation, 8 cells out of 9 showed nuclear fusion (Fig. 3) and simultaneously changed their behavioral character from CNR to wild type when tested in the test solution. When Paramecium cells were incubated in bacterized culture medium with 5 μM H33258, they showed normal growth indicating that H33258 at this concentration does not affect nuclear functioning. No cell division was observed during the experiments.
Fluorescence images of macronuclei after transplantation of a cnrB macronucleus into cnrA cells. (a,b,c) Recipients at 2, 6, and 12 hours after nuclear transplantation. (d) Cells of uninjected 16A1015 at 6 hours after incubation in DNA-specific fluorescent dye H33258. The whole macronucleus of 16B 1104 (cnr B) was transplanted into cells of 16A 1015 (cnr A). Recipients were stood for 1 hour in cell-free culture medium at 25°C then incubated in cell-free culture medium with 5 μM H33258.
Fluorescence images of macronuclei after transplantation of a cnrB macronucleus into cnrA cells. (a,b,c) Recipients at 2, 6, and 12 hours after nuclear transplantation. (d) Cells of uninjected 16A1015 at 6 hours after incubation in DNA-specific fluorescent dye H33258. The whole macronucleus of 16B 1104 (cnr B) was transplanted into cells of 16A 1015 (cnr A). Recipients were stood for 1 hour in cell-free culture medium at 25°C then incubated in cell-free culture medium with 5 μM H33258.
In genetic recombination tests after macronuclear transplantation, recipients were kept for 20 hours in cell-free culture medium at 25°C to induce nuclear fusion. They were grown in fresh culture medium and then divided into 4 subclones in test tubes. The subclones were grown for about 10 fissions and then monitored for both the ability to swim backward in the test solution and the ability to mate with matingreactive testers. As shown in Table 2, three types of transformants were obtained after macronuclear fusion-reorganization: type-1, CNR with E-mating type, indicating the transformation of mating type; type-2, wild type with O-mating type, indicating the transformation of cnrA character; type-3, wild type with E-mating type, indicating the transformation of both cnrA and mating type. In some cases, both type-1 and type-3 were obtained from the same transplanted cell. These results indicate that macronuclear transplantation can be used in somatic nuclear recombination tests.
Ability of the macronucleus to change a recipient cell’s sexual maturity
The ability of the macronucleus to control the expression of sexuality during clonal development was examined using cnrA and cnrB cells as parents for the donor clones. When these two mutants are crossed, it is possible to distinguish their true progeny, which are wild-type cells, from non-conjugant cells or cells that have undergone macronuclear regeneration (MR) by testing for swimming behaviour. Macronuclei from three different stages were used as donors for the transplantation experiments: macronuclear anlagen (donor 1); macronuclei of immature cells (donors 2, 3, 4); and macronuclei of mature cells (donor 5) that had undergone more than 100 fissions. The macronuclear anlagen is known to be a developing nucleus that is undergoing reorganization of chromosomal DNA, including gene amplifications and rearrangements. The macronuclear fusion-reorganization method is summarized in Fig. 1. After nuclear transplantation, the recipient cells (C103) were first incubated in K-DS overnight and then transfered to bacterized fresh culture medium. Two fissions after nuclear transplantation, 4 daughter cells were isolated into capillaries and grown for about 4 fissions (Haga and Hiwatashi, 1981). The mating reactivity of each subclone was assessed by mixing with complementary mating-type cells of a cnrB strain. The mating reactivity of each subclone was determined by the percentage of mating-reactive cells to total cells tested. After testing for the mating reaction, non-reactive cells that showed a wild-type phenotype in swimming behavior were isolated and cloned, and examined with respect to the retention of trans-formed phenotype. When a macronucleus from cells that had undergone about 15 fissions after conjugation was transplanted into a mature cell, at least one subclone derived from each recipient showed loss of mating reactivity within 4 to 10 fissions after transplantation. On the contrary, when the macronucleus from a mature cell was transplanted into an immature cell, all subclones remained in immaturity. As summarized in Table 3, the ability of an immature macronucleus to transform mature cells into change throughout the immaturity period; at the beginning of immaturity, the activity was low or non-existent, and reached a maximum at about 15 fissions. The retention of immaturity induced by the transplantation of an immature macronucleus lasted for about 30 fissions after the transplantation.
Germnuclear activity of the micronucleus in the immaturity period
In normal development, the clonal age of the micronucleus, expressed in terms of the number of replications, is identical to that of the macronucleus. To determine whether the micronucleus changes its ability to undergo meiosis and to produce progeny during the period of sexual immaturity, heterokaryons were produced by macronuclear regeneration (MR) using behavioral markers as described above. Three strains independently produced by MR, HKBab1, 2, and 3, were examined for their micronuclear function by selfing conjugation. As shown in Table 4, all stocks showed the production of true progeny at the age of 15-17 and 25-27 fissions after their selfing conjugations.
DISCUSSION
Sexual immaturity during clonal development of Paramecium is assumed to be controlled by the genes necessary for the synthesis of immaturin, which in turn control the genes for the mating substances (Miwa et al., 1975; Tsukii and Hiwatashi, 1983). The macronuclear reorganization method described here demonstrated that two macronuclei brought together by transplantation are completely mixed and reorganized in recipient cells. An important finding in the present study with regard to the expression of sexual activity is that when immature and mature macronuclei are fused in either immature or mature cells, the sexual activity of the clones derived from the reorganized macronucleus is always immature. Thus the macronucleus in the immaturity period was found to be dominant over the mature macronucleus in terms of the expression of sexual activity.
In the macronucleoplasm-transplantation experiments in P. caudatum, both stable and unstable transformants were obtained in the clones derived from the recipients (Harumoto and Hiwatashi, 1992). In the case of unstable transformation, the transplanted macronucleoplasm was unequally distributed to daughter cells and eventually sorted out. However, the transformation of sexual activity from mature to immature cells is not due to the sorting out of the mature macronucleus as evidenced by the fact that fusion between the donor and recipient’s macronuclei produces a newly reorganized macronucleus that shows the recombination of marker genes (Table 2). In addition, recombinants between mature and immature macronuclei were obtained in the subclones derived from the recipients (Table 3).
Because immature cells cannot mate, it is difficult to determine directly whether the micronucleus can function as a germ nucleus during the immaturity period. The results described here suggest that the micronucleus seems to have no immaturity period because the cells of a heterokaryon whose micronucleus underwent only about 15 divisions produced true progeny by selfing conjugation. However, the possibility cannot be ruled out that the ability of the micronucleus to produce progeny after transplantation in a mature cell is somehow due to the effect of the cytoplasm.
Sexual expression is found to be associated mainly with the clonal age of the macronucleus. Previously, we demonstrated that when purified immaturin is injected into mature cells, the percentage of immature cells brought about by the injection is proportional to the dose of immaturin (Haga and Hiwatashi, 1981). Thus, the percentage of immature clones brought about by the macronuclear reorganization shown in Table 3 should reflect the activity of immaturin production in the reorganized macronucleus.
The ability of the immature macronucleus to render mature cells immature after macronuclear fusionreorganization seems to be related to the number of nuclear divisions after conjugation. This change in macronuclear activity is consistent with the observation reported by Miwa (1984) that the cytoplasmic activity of immaturin present in cells immediately after conjugation is very low. The activity level increases during the next 10 fissions, reaching a maximum at about 15 fissions after conjugation. Analysis of the immaturin effect brought about by microinjection and the retention of this effect in clones after proliferation indicates that after microinjection, immaturin is distributed randomly into the daughter cells and simply diluted during subsequent cell divisions (Haga, 1979). The results suggest that the introduction of immaturin into a cell does not induce the production of immaturin in the recipient. Thus, the production of immaturin at various stages in the immaturity period must be associated with the clonal age of the macronucleus.
Chromosomal DNAs of several species of ciliates exhibit significant differences between micronucleus and macronucleus with respect to DNA contents, sequences, gene alignment, and nucleosome structures (Altschuler and Yao, 1985; Conover and Brunk, 1986; Karrer, 1986; Klobutcher and Prescott, 1986; Pan and Blackburn, 1981; Preer and Preer, 1979; Tao and Yao, 1981). However, it is unknown whether macronuclear genomic DNAs differ between sexually immature and mature cells. Preliminary studies show that the microinjection of genomic DNA isolated from young immature cells renders mature cells immature for about 20 fissions after the microinjection. The effect was also induced by the microinjection of NotI-digested DNA fragments but not induced by EcoRI-digested DNA fragments. This indicates that specific DNA sequences are involved in the transformation of sexuality (N. Haga, unpublished observation).
It has been demonstrated in P. tetraurelia that the clonal age required for autogamy was shortened by repeated elimination of a part of the macronucleus (Mikami and Koizumi, 1983). Based on this finding, it was suggested that the clonal age of Paramecium should be measured in terms of rounds of chromosome replication or DNA synthesis rather than in terms of cell divisions. If the macronucleus has the ability to count the number of replications, and thus decide the timing of immaturin gene expression, the present line of research should help to provide an understanding of the somatic nucleus as a developmental clock controlling the intrinsic temporal programming of aging.
ACKNOWLEDGEMENTS
The author thanks K. Hiwatashi and D. Cronkite for their critical reading of the manuscript and valuable comments. This research was supported in part by grants from the Japan Ministry of Education, Science and Culture (Grant-in-Aid for Cooperative Research 03304001), and the Narishige Zoological Science Award.