Induction of autogamy by transfer of macronuclear karyoplasm in Paramecium tetraurelia

Macronuclear karyoplasm was transplanted from pre-autogamous donor cells (clonal age, 22 fissions) into the macronucleus of young recipient cells (2 fissions after autogamy occurred) by means of microinjection. A reciprocal experiment was carried out by injecting karyoplasm from young clonal age donors into pre-autogamous recipients. In the case of karyoplasm transfer from pre-autogamous donors to young recipients, autogamy occurred early in 67% of injected cells, whereas reciprocal injections had no influence on the onset of autogamy, and all of the injected cells underwent autogamy. Such results indicate a distinct role of pre-autogamous cells of macronucleus in the induction of autogamy.     

A Study of Odd Mating-Type Determination Factor by Transfer of Macronuclear Karyoplasm in PARAMECIUM TETRAURELIA

The unique feature of the "B system" of mating-type determination found in Paramecium tetraurelia is the existence of a cytoplasmic difference between odd (O) and even (E) cells created and maintained by the action of their macronuclei. Thus far, the presence of a determining factor that controls the differentiation of the developing zygotic macronucleus for O mating type has not been verified. Results of crosses between cells of differing clonal age and complementary mating type suggest that, for one to two fissions after autogamy, O cells produce some factor that determines the gametic nucleus (micronucleus) as mating type O. Direct evidence for the production of O-determining factor by the young O macronucleus was obtained by transplanting young O macronuclear karyoplasm (a part of the macronucleus) into E cells: 32-35% of E exautogamous clones transformed to O; transformation of E exautogamous clones to O reached as high as 72% by transfer of young O macronuclear karyoplasm from a conjugant, 3-4 hr after mixing. This indicates that O determinants produced by the O macronucleus can also act during the sensitive period of development of the new macronucleus. These O-determining factors may be produced or activated at the sexual stage and then decrease in activity in subsequent fissions after new macronuclear reorganization.     

Nuclear transplant studies of the determination of mating type in germ nuclei of Paramecium tetraurelia

The micronucleus from vegetative cells of one mating type (O or E) in Paramecium tetraurelia was transplanted by micropipet into amicronucleate cells of opposite mating type (E or O). When autogamy was induced in the recipient cells, they developed new macronuclei and micronuclei derived from the transplanted micronucleus and usually expressed the same mating type as the recipients. The results indicate that micronuclei in the asexual phase may be undetermined for mating type. Recipient E cells in which the macronucleus had been previously removed were transplanted with a whole macronucleus from an O cell. Their mating type was soon transformed E to O before the occurrence of autogamy, and remained O after autogamy. This demonstrates that the transplanted macronucleus determined the O cytoplasmic state to determine the developing zygotic macronucleus for mating type O. It is unlikely that the micronucleus is determined for mating type in O or E cell during the asexual cycle.     

Trichocyst phenotype transformation induced by macronuclear transplantation in Paramecium tetraurelia

A portion of the macronucleus of wild-type cells of Paramecium tetraurelia was removed and was injected into cells homozygous for the ftA mutation. The ftA mutants make defective trichocysts and are unable to perform normal trichocyst exocytosis. After injection, approx. 30% of the surviving cells show a phenotype shift from mutant to wild-type. This shift is stable during subsequent vegetative growth until clonal death. If, however, the hybrid cell lines are brought to autogamy (which discards the existing macronucleus and forms a new one from sexual products derived from a micronucleus), then the lines revert to the ftA phenotype. Since micronuclei were not transplanted, the phenotypic reversion after autogamy is to be expected, and demonstrates that the transformation affects the macronucleus only. A second series of injections involved transfer of a portion of the macronucleus from cells homozygous for the trichocyst ptA mutation into ftA host cells. These two mutations are genetically complementary, so the injection should be genetically equivalent to forming a double heterozygote. Approx. 20% of the injection survivors shift to wild-type. This shift is also vegetatively stable unless autogamy occurs; after autogamy, reversion to the ftA phenotype is seen. These results show that a portion of a macronucleus can be successfully transplanted from one cell to another and that, in the host cytoplasmic environment, normal gene expression and replication of a transplanted macronucleus does occur. The technique of macronuclear transplantation is significant to studies of the macronuclear contribution to clonal aging, and to studies on genetic control over trichocyst development.     

Clonal Age and the Proportion of Defective Progeny after Autogamy in PARAMECIUM AURELIA

The relation of mortality and the proportion of progeny with reduced fission after autogamy to the clonal age in Paramecium aurelia was investigated. This relation is not linear but the proportion of defective progeny increases stepwise. The observations are in agreement with those expected from the calculations of the number of deleterious mutations in the micronucleus.     

Local Degeneration of Cilia and Nuclear Activation during Sexual Reproduction in Paramecium caudatum

The correlation between local degeneration of cilia and nuclear activation during chemically induced autogamy in Paramecium caudatum was studied by experimental interruption of autogamy. When the induction of autogamy was interrupted 60 min after the onset of chemical induction, nuclear activation did not occur and ciliary regeneration took place at the deciliated surface. On the contrary, when it was interrupted 3 hr after the onset of induction, nuclear activation was not stopped and no reciliation was observed. At this critical stage, the deciliated zone was seen to extend to the right wall of the gullet. These findings suggest that there is a transition point in activation processes. It is conceivable that in autogamous cells that have gone beyond this transition point all processes of nuclear activation proceed, and that the ability to regenerate cilia is lost by the end of autogamy, even if the stimulus for autogamy is removed.     

Microinjection of plasmid DNA encoding the A surface antigen of Paramecium tetraurelia restores the ability to regenerate a wild-type macronucleus.

Strain d48 of Paramecium tetraurelia contains the A i-antigen gene in the micronucleus, but the gene is lost when micronuclear products develop into the macronucleus. It has recently been shown that when injected into d48, macronucleoplasm from the wild type transforms d48 cells to wild type. It is shown here that wild-type cytoplasm can also bring about transformation, with a marked stage-specific sensitivity for both donor and recipient. It was also found that a plasmid containing the cloned A gene could transform d48 to wild type. Injection of nucleoplasm from animals in the vegetative stage of the cell cycle into the cytoplasm of recipients at various stages of autogamy caused high-frequency transformation of cells able to express the A serotype both before and after the next autogamy. Injection of nucleoplasm into vegetative macronuclei produced over 70% transformants able to express the A serotype after the next autogamy. The ability of nucleoplasm to transform was acquired at the second cell cycle after autogamy and was maintained throughout the vegetative stage. When cytoplasm was obtained from donors during autogamy and injected into the cytoplasm of recipients 1 to 2 h after the sensitive period, quite high frequencies of stable revertants were found when tested both before and after the next autogamy. Cells that were injected into the macronucleus with the cloned A plasmid expressed the A serotype after five fissions in over 20% of the lines and maintained this ability through successive fissions; all transformants except one stably expressed the A serotype even after the next autogamy.     

Commitment to autogamy in Paramecium blocks mating reactivity: implications for regulation of the sexual pathway and the breeding system

Commitment to autogamy blocks mating reactivity in Paramecium. Cells which had previously developed mating reactivity, lost reactivity 30-90 min prior to the preautogamous fission. Mating reactivity develops at a standard level of starvation when cells are allowed to exhaust their food supply naturally. In abruptly starved cultures, mating reactivity appears 3.3 h after downshift. Autogamy is also triggered by starvation. The level of starvation required for initiation of autogamy decreases progressively as cells age. When the autogamy starvation threshold drops to such a low level that all cells become committed to autogamy before any of them develop mating reactivity, reactivity does not occur under natural starvation conditions and the period of maturity for conjugation has come to an end. There is no absolute immature period for autogamy.     

Nuclear Roles in the Post-Conjugant Development of the Ciliate Euplotes aediculatus II. Experimentally Induced Regeneration of Old Macronuclear Fragments1

Following conjugation in ciliates, the usual fate of the old pre-conjugant macronucleus is resorption. In some species, however, old macronuclei, or their fragments, have the ability to reform functional vegetative macronuclei when new macronuclear anlagen are defective. The present work on Euplotes shows that if anlagen are allowed to carry out their essential roles in early exconjugant development, including influence on cortical reorganization such that feeding can resume, they can then be permanently damaged by UV-microbeam irradiation and regeneration of old macronuclear fragments can occur. E. aediculatus exconjugants were anlage-irradiated at 40–60 hr of development and the irradiated cells cultured individually and fed. Squashes revealed enlargement and anteriorward migration of the persistent (posterior) macronuclear fragments. The first post-conjugant fission of such cells was delayed (times ranged 6–43 days) and did not seem to involve the damaged anlagen, which remained rudimentary, did not divide along with the cells, and were subsequently resorbed. It appeared that cell fission was supported by the fragments of the old macronuclei, which either divided or partitioned themselves between the two daughter cells. Mating tests performed on early clones derived from irradiated exconjugants revealed ample conjugation competence; intraclonal conjugation in such clones was also apparent. The absence of the immature period seen in normal exconjugants provides further evidence that the clones arose from cells with regenerated macronuclei.     

Behavior of germinal micronuclei under control of the somatic macronucleus during conjugation in Paramecium caudatum.

In conjugating pairs of Paramecium caudatum, the micronuclear events occur synchronously in both members of the pair. To find out whether micronuclear behavior is controlled by the somatic macronucleus or by the germinal micronucleus, and whether or not synchronization of micronuclear behavior is due to intercellular communication between conjugating cells, the behavior of the micronucleus was examined after removal of the macronuclei from either or both cells of a mating pair at various stages of conjugation. When macronuclei were removed from both cells of a pair, micronuclear development was arrested 1 to 1.5 hr after macronuclear removal. When the macronucleus of a micronucleate cell mating with an amicronucleate cell was removed later than 3 to 3.5 hr of conjugation, that is, an early stage of meiotic prophase of the micronucleus, micronuclear events occurred normally in the operated cell. These results suggest that most micronuclear events are under the control of the macronucleus and that the gene products provided by the macronucleus are transferable between mating cells. One such product is required for induction of micronuclear division and is provided just before metaphase of the first meiotic division of the micronucleus. This factor is effective at a lower concentration in the cytoplasm and/or is more transferable between mating cells than the factors required for other stages. This factor, which seems to be present at least until the stage of micronuclear disintegration, is able to induce repeated micronuclear division as long as it remains active. The factor can act on a micronucleus which has not passed through a meiotic prophase. Moreover, the results suggest the existence of a second factor which is provided by the macronucleus after the first meiotic division that inhibits further micronuclear division.     

Nuclear Roles in the Post-Conjugant Development of the Ciliate Euplotes aediculatus. III. Roles of Old Macronuclear Fragments in Nuclear and Cortical Development1

Following conjugation of the hypotrichous ciliate Euplotes aediculatus, the posterior fragments of the old (prezygotic) macronucleus persist until after the first vegetative division. These fragments remain viable during exconjugant development as shown by their ability to regenerate should the cell's new macronucleus be damaged. It thus seemed possible that these parental nuclear fragments might participate in the development of the new macronucleus and/or the crucial post-conjugant cortical reorganization that restores the exconjugant cell's ability to feed. This idea was tested by damaging the posterior fragments with various doses of microbeam ultraviolet (UV) light and assessing the results of such treatment on subsequent cortical and nuclear development. When the posterior fragments of the macronucleus were irradiated at the beginning of cortical morphogenesis, the new macronucleus in 1/3 to 1/2 of the cells assumed a “folded” appearance but did not mature. These cells did not undergo cortical reorganization. Cells irradiated at earlier stages did not detectably develop an oral apparatus; their new macronucleus remained arrested at the spherical anlage stage. The results show that the posterior fragments of the parental macronucleus are necessary for normal nuclear and cortical development. These old nuclear fragments appear to influence the growing macronuclear anlage directly and probably the cortex as well. There also appears to be an information flow from the non-irradiated partner of a persistently joined exconjugant doublet to its irradiated counterpart, enabling normal anlage and cortex development in the irradiated cell.     

Effects of K+ and the K+ Ionophore Valinomycin on the Post-Conjugational Nuclear Differentiation of Paramecium caudatum

For determination of the effect of K⁺ on macro- and micronuclear differentiation Paramecium caudatum exconjugants were transferred to medium with various concentrations of Valinomycin and/or K⁺ at the critical stage of nuclear differentiation. The differentiation was not disturbed by transfer to medium containing 1.5 mM to 50 mM KCl. Injection of KCl solution at the critical stage also did not affect differentiation of the macronucleus appreciably. But change of the KCl concentration in the medium at the critical stage interrupted of normal development of the macronucleus.
Macro- and micronuclear differentiations after conjugation are known to be determined by the antero-posterior localization of postzygotic micronuclei. This nuclear localization is achieved by elongation of mitotic spindles and marked shortening of the cell length at the time of micronuclear division. Successive measurements of cell length at 25°C showed that cells began to shorten 1.5 hr after mating-pair separation, reaching to half the initial length about 2.5 hr after the separation, and then returning to recover their initial length within about 50 min. In a solution of K⁺ (50 mM) plus Valinomycin (1μg/ml or more), cell shortening was inhibited. It is not known whether elongation of mitotic spindles at the time of critical nuclear division was disordered by this treatment, but the macronuclear anlagen developed in the treated cells. Thus shortening in the cell length is not indispensable for nuclear differentiation.     

Interactions between newly developed macronuclei and maternal macronuclei in sexually immature multinucleate exconjugants of Paramecium caudatum

The macronucleus of Paramecium caudatum controls most cellular activities, including sexual immaturity after conjugation. Exconjugant cells have two macronuclear forms: (1) fragments of the maternal macronucleus, and (2) the new macronuclei that develop from the division products of a fertilization micronucleus. The fragments are distributed into daughter cells without nuclear division and persist for at least eight cell cycles after conjugation. Conjugation between heterokaryons revealed that the fragmented maternal macronuclei continued to express genetic information for up to eight cell cycles. When the newly developed macronucleus was removed artificially within four cell cycles after conjugation, the clones regenerated the macronuclear fragments (macronuclear regeneration; MR) and showed mating reactivity, because they were sexually mature. However, when the new macronucleus was removed during later stages, many MR clones did not show mating reactivity. In some extreme cases, immaturity continued for more than 50 fissions after conjugation, as seen with normal clones that had new macronuclei derived from a fertilization micronucleus. These results indicate that the immaturity determined by the new macronucleus is not annulled by the regenerated maternal macronucleus. Mature macronuclear fragments may be "reprogrammed" in the presence of the new macronucleus, resulting in their expression of "immaturity."     

Nuclear functions in postconjugational development of Paramecium caudatum: Ability to form food vacuoles analyzed by nuclear elimination and implantation

Paramecium caudatum loses the ability to form food vacuoles at the crescent stage of the micronucleus from 5 to 6 hr after the initiation of conjugation and regains it immediately after the third division of the zygotic nucleus. To assess the micronuclear function in the development of the oral apparatus after coniugation, prezygotic micronuclei was removed from cells at various stages of conjugation, and their ability to form food vacuoles were examined. (1) When all of the prezygotic micronuclear derivatives were eliminated before the stage of formation of the zygotic nucleus, the exconjugant did not regain its ability. (2) When a zygotic nucleus or postzygotic nuclei were removed, in some cases the cell formed as many food vacuoles as did nonoperated cells after conjugation, while in other operated cells the number of food vacuoles was subnormal. (3) When a micronucleus from a cell at vegetative phase (G1) was transplanted into a cell of an amicronucleate mating pair at the stage between 8 and 9 hr after the initiation of conjugation, the implanted cell regained the ability to form food vacuoles. However, no cell regained the ability when the implantation was carried out within 1 hr after the separation of the mates. The results show that the micronucleus plays an indispensable role in the development of the oral apparatus at the stages of exchange of gametic nuclei and fertilization and that the micronucleus transplanted from asexual cells can fulfill this function. On the other hand, removal of the macronucleus from exconjugants showed that the maternal macronucleus also has an indispensable function in regaining the ability to form food Vacuoles. © 1992 Wiley-Liss, Inc.     

Autogamy in Paramecium cell cycle stage-specific commitment to meiosis

Autogamy is a process of meiosis and fertilization which takes place in unpaired Paramecium cells, and which is triggered by starvation. This study examines the consequences of nutritional down-shift at various points within the cell cycle on the occurrence of autogamy. It shows that cells become committed to autogamy in a two-step process. An initial point of commitment to autogamy occurs about 100 min prior to the median time of cell division (cell cycle duration, 330 min). Cells which have become committed to autogamy initiate meiosis following the next fission, others complete another vegetative cell cycle before undergoing meiosis. Treatments that perturb the cell cycle and displace the point of commitment to division also displace the point of initial commitment to autogamy to the same extent.The initial commitment to autogamy can be reversed by refeeding. The second, final, point of commitment to autogamy occurs about 30 min after the fission, immediately prior to initiation of meiosis, and coincides with the beginning of meiosis. If cells are refed at this point, or at later stages, autogamy continues.Autogamy is not well synchronized either in naturally starved cultures or in those subjected to abrupt nutritional down-shift. This is a consequence of the cell cycle stage dependence of entry into autogamy. Autogamy occurs synchronously in samples of dividers selected from asynchronous cultures 2 or more hours after nutritional down-shift. The timing of the events of conjugation and autogamy coincide when the pre-autogamous fission is aligned temporally with the initial contact of mating cells.     

Induced change in a non-mendelian determinant by transplantation of macronucleoplasm in Paramecium tetraurelia.

Mutant strain d48 of Paramecium tetraurelia lacks the gene for antigen A in the macronucleus, whereas this gene is present in the micronucleus. Transfer of macronucleoplasm from the wild type to strain d48 caused d48 to revert to the wild type after autogamy. Transfer of cytoplasm was not as effective as transfer of macronucleoplasm. It was also found that the micronucleus of d48 developed normally when it was transplanted to wild-type cells, whereas the micronucleus of the wild type formed a macronucleus that lacked the antigen A gene when this micronucleus was transplanted into d48. It was concluded that the micronucleus of d48 has a normal antigen A gene and that the hereditary determinants responsible for the d48 trait are located in the macronucleus. Molecular analysis of d48 clones that had been induced to revert to the wild type revealed that they possessed the antigen A gene in the macronucleus.     

Mutation affecting cell separation and macronuclear resorption during conjugation in Tetrahymena thermophila: early expression of the zygotic genotype.

A new recessive conjugation lethal mutation was found in Tetrahymena thermophila which was named mra for macronuclear resorption arrest. Other events affected by the mra mutations are separation of pairs, DNA replication in the macronuclear anlagen, and resorption of one of the two micronuclei. In wild-type crosses 50% of the pairs had separated by 12 hr after mixing two mating types and had completed resorption of the old macronucleus 1-2 hr later. In contrast most mra conjugants did not separate even by 24 hr after mixing and the old relic (condensed) macronucleus was seen in over 90% of them. After addition of 10 mM calcium to the conjugation medium, the mra conjugants did separate but they still failed to complete resorption of the old macronucleus and to replicate macronuclear anlagen DNA in the exconjugants. The calcium induced separation of the mra conjugants occurred later than the separation of control pairs. During normal conjugation cell separation occurs before the first expression of known macronuclear genes and prior to processing of the macronuclear DNA. Therefore, the mra phenotype infers that separation of conjugants requires a signal which is produced by the macronuclear anlagen at an unusually early time.     

DNA synthesis following gross alterations of the nucleocytoplasmic ratio in the ciliate Stentor coeruleus

Incorporation of externally supplied and injected ³H-thymidine into DNA was measured autoradiographically. Starved stentors synthesized no DNA, in contrast to well-fed animals, but replication commenced in some cases if they were fed. Grafting starved and well-fed stentors together rapidly induced DNA synthesis in the starved partner. Suppression of synthesis in the well-fed macronucleus was not observed. Well-fed cytoplasm alone induced DNA synthesis in starved stentors, and starved cytoplasm grafted to starved animals also induced synthesis after a lag. Starved animals with the beaded macronucleus reduced to 2 nodes commenced DNA replication after 6 hr; however, initiation was prevented if the normal nuclear complement was restored before the fourth hour.The macronucleus was required to render starved cytoplasm capable of supporting DNA synthesis, but once potentiated the cytoplasm alone could initiate replication in a starved nucleus. Initiation required RNA synthesis, shown by actinomycin sensitivity.This nucleic acid analysis suggests that decreasing the nucleocytoplasmic ratio elicits RNA synthesis in the remaining macronucleus. The RNA codes for proteins involved in DNA synthesis which are synthesized in the cytoplasm and enter the nucleus to initiate DNA replication.     

Endocrine control of the metamorphosis of the larval muscles in Calliphora erythrocephala (diptera): In vitro studies of the role of ecdysteroids

The larval muscle fibers of Calliphora can be divided into two categories according to their evolution during metamorphosis; in the major category, the fibers degenerate completely and disappear, whereas in the other they undergo partial involution and differentiate into adult fibers. Muscles belonging to these two categories were excised at various times during the last larval instar and cultured in vitro in the presence or absence of synthetic moulting hormones; morphological changes were investigated by electron microscopy. In hormone-free medium, a critical period was observed about 16 hr before pupariation; irrespective of their category, fibers excised before this period showed no morphological changes, whereas fibers excised after the critical period exhibited modifications similar to those observed in vivo. In medium containing 20-hydroxyecdysone excised fibers which would degenerate in vivo undergo the same process, regardless of the moment of excision. Conversely, fibers destined to persist in vivo in the imago, develop differently in the presence of 20-hydroxyecdysone according to the time of excision. When excised before the critical period, they show the same signs of degeneration as fibers destined to disappear in vivo, whereas when excised after the critical period, they exhibit only an involution of their contractile elements comparable to that observed in normal fibers, in vivo, prior to their redifferentiation.