Nuclear Behavior During Reconjugation in Euplotes patella (Ciliophora,Hypotrichida)1

Nuclear behavior during reconjugation and the ultimate fate of the ex-reconjugants were followed after induction of reconjugation in Euplotes patella. An exconjugant could reconjugate with a vegetative cell or with another exconjugant. Exconjugants at an early stage of macronuclear development (oval macronuclear anlagen) did not reconjugate frequently whereas exconjugants at a late stage of macronuclear development (rod-like macronuclear anlagen) reconjugated frequently. In all cases, the micronucleus underwent normal meiosis and other nuclear changes. After reconjugation, a new macronuclear anlage and a new micronucleus were formed normally, so that there were two kinds of macronuclear anlagen in the exconjugants, an old and a new. The old rod-shaped anlage did not disappear after the differentiation of a new one, but it was broken up into several fragments. While the survival rate after normal conjugation was 78%, it was 0–20% after reconjugation. These results suggest that the micronuclei of exconjugants can act as germ nuclei even at a very early stage and that reconjugation, unlike conjugation, is harmful to the cell.     

Nuclear Behavior of Euplotes woodruffi During Conjugation

SYNOPSIS. During conjugation of E. woodruffi , the micro-nucleus divides repeatedly four times prior to synkaryon formation and twice thereafter. The first division resembles an ordinary somatic mitosis, resulting in the formation of two daughter nuclei in each conjugant. Both products of this division enter the second division which corresponds to the heterotypic division of other ciliates, characterized by a parachute stage. Following this stage sixteen bivalents appear and separate into dyads and pass to the poles. During the following divisions individualized chromosomes do not appear but only certain chromatin elements comparable to those seen in the somatic and preliminary divisions. These divide and pass to the poles. All daughter nuclei of the second division enter and complete the third division. Only two of the products of the third division enter the final pregamic division while the rest degenerate. Exchange of pronuclei and their fusion leads to synkaryon formation. The conjugants then separate and in each exconjugant the synkaryon divides twice in rapid succession. Of the four products one condenses to become the functional micronucleus, another enlarges rapidly to become the macronuclear anlage while the remaining two degenerate and disintegrate. The old macronucleus breaks into irregular and polymorphic bodies. As the macronuclear anlage enlarges the remnants of the old macronucleus reorganize and fuse with the macronuclear anlage to form a characteristic vegetative macronucleus.     

Genomic exclusion in Tetrahymena thermophila: A cytogenetic and cytofluorimetric study

Genomic exclusion is an aberrant form of conjugation of Tetrahymena thermophila in which the genome of a defective conjugant is excluded from the genotype of the exconjugant progeny. This paper is concerned with the cytogenetic and nucleocytoplasmic events of genomic exclusion in senescent clones A*III and C*. In crosses between A*III or C* and strain B, functional, haploid gametic nuclei are formed only in the strain B cell. In some instances one of the gametic nuclei divides prior to transfer of the migratory gametic nucleus, and both products then undergo DNA synthesis. Two alternative cytogenetic pathways are followed after transfer of the migratory nucleus. In the first, the conjugants separate without further micronuclear divisions. This pathway was most common in A*III genomic exclusion. In exconjugants the former gametic nuclei undergo both DNA synthesis and (presumably) intranuclear separation of centromeres to restore micronuclear diploidy. The old macronucleus of each exconjugant is retained without autolysis. This class of exconjugant survives and contributes genes to future sexual progeny. In the second cytogenetic pathway the gametic nuclei divide and macronuclear anlagen are formed, as in normal conjugation. This pathway was more common in C* genomic exclusion. The initial DNA content of the anlagen ranges from haploid to diploid. Following two to three rounds of DNA synthesis, further macronuclear development ceases and the anlagen appear to undergo autolysis. The old macronucleus condenses and also undergoes autolysis, as in normal conjugation. Except for rare C* exconjugants, in which macronuclear development is completed, anlagen-bearing genomic exclusion exconjugants die. Death may be caused by aneuploidy, errors in the timing or receptivity to signals for autolysis, or the inability of anlagen-bearing exconjugants to feed. Anlagenbearing conjugants are frequently abnormal with respect to the number of anlagen and micronuclei. Most of the anomalies can be explained by postulating errors in the timing of both developmental signals and nuclear divisions. Rare conjugants in which gametic nuclei divide but do not give rise to macronuclear anlagen are also observed. In these instances, the old macronuclei condense and undergo autolysis. Destruction of the old macronucleus therefore is independent of the presence of macronuclear anlagen and requires cell pairing in order to be initiated.     

Developmental expression of macronuclear specific antigen in Paramecium caudatum.

We obtained a monoclonal antibody (MA-1) specific for macronuclei of the ciliate Paramecium caudatum and P. dubosqui. Immunoblotting showed that the antigen was a polypeptide of 50 kilodalton (kDa). During the process of nuclear differentiation in P. caudatum, the MA-1 antigens appeared in the macronuclear anlagen immediately after four out of eight post zygotic nuclei differentiated morphologically into the macronuclear anlagen. Afterwards, the antigens could be detected in the macronucleus through the cell cycle, and disappeared when the macronucleus began to degenerate in exconjugant cells. These results suggest that the antigens may play a role in the differentiation and function of the macronucleus.     

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.     

Macronuclear differentiation in conjugating pairs of Tetrahymena treated with the antitubulin drug nocodazole

During Tetrahymena conjugation gamic nuclei (pronuclei) are produced, reciprocally exchanged, and fused in each mate. The synkaryon divides twice; the two anterior nuclei develop into new macronuclei while the two posterior nuclei become micronuclei. The postzygotic divisions were blocked with the antitubulin drug nocodazole (ND). Then pronuclei (gamic nuclei) developed directly into macronuclear anlagen (primordial macronuclei), inducing amicronucleate cells with two anlagen, or, rarely, cells with one anlagen and one micronucleus. ND had a similar effect on cells that passed the first postzygotic division inducing amicronucleate cells with two anlagen, while cells treated with ND at the synkarya stage produced only one large anlage. Different intracytoplasmic positioning of the nuclei treated with ND (pronuclei, synkarya and two products of the first division) shows that most of cell cytoplasm is competent for inducing macronuclear development. Only posteriorly positioned nuclei--products of the second postzygotic division--remain micronuclei. The total cell DNA content, measured cytophotometrically in control and in ND-induced amicronucleate conjugant cells with one and two anlagen, was similar in all three samples at 12 h of conjugation. Eventually, at 24 h this content was about 2 pg (8 C) per anlagen both in nonrefed control and in amicronucleate exconjugants. Therefore "large" nuclei developing in the presence of ND were true macronuclear anlagen.     

Positional control of nuclear differentiation in paramecium

Nuclear reorganization, which results in the differentiation between macronuclear anlagen and micronuclei during autogamy or conjugation in Paramecium tetraurelia, was compared in wild-type cells and in two mutants, mic44 and kin241, which form abnormal numbers of macronuclear anlagen and micronuclei. Our observations show that all macronuclear anlagen derive from the nuclei positioned at the posterior pole of the cell at the second postzygotic division. This posterior localization is transient and correlated with a marked change in cell shape and decrease of cell length. These results suggest that cytoplasmic or cortical factors precisely located in the posterior pole are essential to trigger macronuclear differentiation and that the control of nuclear positioning is dependent upon precise modifications of cell shape.     

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.     

Maternal effect mutations affecting macronuclear determination and development in Paramecium tetraurelia

Gene mutations that interfere with macronuclear development in Paramecium were obtained by selecting lines that failed to produce normal macronuclear anlagen following the second autogamy after mutagenesis. The mutants fell into several complementation groups. There was one case of apparent intragenic noncomplementation among the eight mutants examined. In the stronger mutants, macronuclear anlagen were not formed, and all four mitotic products of the posfzygotic divisions of the synkaryon remained as micronuclei. Under semirestrictive conditions, cells often contained a single anlage, suggesting that determination of anlagen was a discrete event for each nucleus. The missing anlagen trait was recessive and associated with a strong maternal effect. The phenocritical period of one of the stronger alleles, aal^a, began at the second postzygotic division and ended with the first morphological differentiation of macronuclear anlagen. Nuclear migration in this mutant was abnormal. Under restrictive conditions, the posterior products of the second postzygotic division reached a posterior-most position, which was 8% of cell length more anterior than that of the most posterior nuclei in wild-type cells. Under permissive conditions, the pattern of migration was intermediate between that of wild-type cells and mutants under fully restrictive conditions. The patterns of nuclear migration were consistent with the nuclear growth kinetics.     

Disturbance of the determination of germinal and somatic nuclei by heat shock in Paramecium caudatum

During conjugation of Paramecium caudatum, nuclear determination occurs soon after the third postzygotic division: one of the four anterior nuclei becomes the micronucleus and the remaining three degenerate, while four posterior nuclei differentiate into macronuclear anlagen. Macronuclear differentiation is supposed to be dependent on a cytoplasmic differentiation factor. In this study, postzygotic cells were subjected to heat shock for 30 min and nuclear changes were observed by staining with carbol fuchsin solution. When heat shock was initiated during the period from metaphase to telophase of the third postzygotic division, cells showed an excess of macronuclear anlagen and were typically amicronucleate. Abnormal nuclear localization around the end of the third (last) postzygotic division may explain the origin of these kinds of cells. A similar phenomenon appeared after treatment with actinomycin D or emetine. Since heat shock did not inhibit macronuclear differentiation but destroyed the formation of micronuclei, some factor(s) probably plays an essential role in nuclear determination, especially in the protection of the micronuclei.     

Nuclear Behavior of Spirostomum ambiguum During Conjugation with Special Reference to Macronuclear Development*

SYNOPSIS. During conjugation in Spirostomum ambiguum, the micronuclei divide thrice before synkaryon formation and 20 times thereafter. During the first meiotic division 18-24 bivalents, each about 0.5 μ or less appear on the spindle. They separate and pass to the poles. The details of the 2nd and 3rd prezygotic divisions and synkaryon formation by reciprocal exchange of gametic nuclei resemble those described for other ciliates in the literature. The synkaryon divides twice resulting in 4 nuclei; 2 of them become micronuclei and the remaining 2 macronuclear anlagen. The micronuclei enter into division, but this division is arrested in metaphase. The chromosomes in the macronuclear anlagen resemble those appearing in the Ist meiotic division in shape and size. In their maximum stage of development the macronuclear chromosomes are at least 3-4 times larger than those appearing in the arrested micronuclear metaphases in the same cell. There is no banding pattern of the chromosomes and therefore the possible extent of polyteny is difficult to evaluate. The chromosomes duplicate 3-4 times resulting in about 200–250 before they become indistinct as separate entities. Spirostomum is the only nonhypotrichous ciliate in which these cytologic features are described.     

Macronuclear Regeneration and Cell Division in Paramecium caudatum

SYNOPSIS. In Paramecium caudatum , occurrence of macronuclear regeneration is closely related to the time of feeding after conjugation. Macronuclear regeneration is induced with a high frequency when conjugating pairs are transferred into fresh culture medium. Feeding immediately after conjugation induces early cell division and 3 or more fissions occur without macronuclear division because of the inability of the macronuclear anlagen to divide. In the cells lacking normal macronuclear anlagen, old macronuclear fragments undergo regeneration and form vegetative macronuclei.     

Autogamy in Paramecium polycaryum

Mass cultures of a stock of Paramecium polycaryum maintained over a period of several years showed abundant and frequent nuclear reorganization stages resembling those of ex-conjugant and ex-autogamous animals of other species of Paramecium. Conjugation has never been reported for P. polycaryum, nor has it been found in these studies. Cytological examination of stained preparations revealed a process of autogamy in P. polycaryum, closely similar to that described previously for P. aurelia. As a rule, all four of the micronuclei, the typical vegetative number in P. polycaryum, engage in the first prezygotic division which is characterized by the formation of prophase crescents. Variable numbers of the eight nuclei continue with the second division. A maximum of sixteen nuclei may result. Apparently, only one of these normally completes the third prezygotic division to form the gametic nuclei, although more than one may initiate it. A fusion nucleus (synkaryon) arises in, or near, a paroral cone, thus paralleling autogamy in P. aurelia. A series of postzygotic divisions produces eight definitive nuclei, four of which become macronuclear anlagen and four remain micronuclei. The first division of the synkaryon results, possibly, in the formation of a viable nucleus and a non-viable one, as in ex-conjugants of P. caudatum. After the last micronuclear division, a skein evolves from the old macronucleus which has become flattened and leaf-like. The skein rapidly segments into "sausages" which transform into spherical fragments, about thirty in number. Two cell divisions restore the normal vegetative nuclear complex.     

Developmental expression of macronuclear specific antigen in Paramecium caudatum

We obtained a monoclonal antibody (MA-1) specific for macronuclei of the ciliate Paramecium caudotum and P. dubosqui. Immunoblotting showed that the antigen was a poly-peptide of 50 kilodalton (kDa). During the process of nuclear differentiation in P. caudatum, the MA-1 antigens appeared in the macronuclear anlagen immediately after four out of eight post zygotic nuclei differentiated morphologically into the macro-nuclear anlagen. Afterwards, the antigens could be detected in the macronucleus through the cell cycle, and disappeared when the macronucleus began to degenerate in exconjugant cells. These results suggest that the antigens may play a role in the differentiation and function of the macronucleus. © 1992 Wiley-Liss, Inc.     

Observations on the Life-cycle of a New Race of Blepharisma undulans from India

SYNOPSIS. A full account of the nuclear changes during binary fission and conjugation in a local race of Blepharisma is presented in this paper. The macronucleus consists of 2 nodes connected by a strand. Number of micronuclei varies from 6 to 18. During binary fission, condensation of macronucleus is followed by elongation and thinning of the middle region which finally breaks. Daughter nuclei later attain the typical vegetative form. Notably, during binary fission some micronuclei appear to complete their mitoses by the time the macronucleus attains the condensed form, while others lag behind and exhibit practically every stage of mitosis.
During conjugation, from 6 to 10 micronuclei undergo the first pregamic division, the same number through the second division, and two products of the second division take part in the third division. The rest degenerate. Division products of the nuclei in the paraoral region take part in synkaryon formation. The synkaryon undergoes either 2 or 3 divisions. In the former case, of the 4 products, 2 become the macronuclear anlagen, one the micronucleus and the fourth degenerates. In the latter case, of the 8 products, 3 to 4 become the macronuclear anlagen and the rest become micronuclei. Chromatin elimination has been observed during the division of the macronuclear anlage, followed by an extra metagamic fission of the cell.
Comparison with two other races from India and an American race indicates considerable diversity in the structure and behaviour of the nuclear apparatus in different races of Blepharisma undulans.     

Dynamic behaviour of stationary pronuclei during their positioning in Paramecium caudatum

During conjugation of Paramecium caudatum, there are two well-known stages when nuclear migration occurs. What happens to the nuclei is closely related to their localisations in cells. The first of these stages is the entrance of one meiotic product into the paroral region. This nucleus survives, while the remaining three outside this area degenerate. The second stage is the antero-posterior localisation of eight synkaryon division products. Four posterior nuclei are differentiated into macronuclear anlagen, whereas four anterior nuclei remain as the presumptive micronuclei. In this experiment, the process of the third prezygotic division of P. caudatum was studied with the help of protargol staining. Here, a third nuclear migration was discovered. By two spindle turnings and two spindle elongations, stationary pronuclei were positioned near migratory pronuclei. This positioning of stationary pronuclei could shorten the distance for transferred migratory pronuclei to recognise and reach the stationary pronuclei. This fosters the synkaryon formation of P. caudatum.     

Nuclear Differentiation in Paramecium caudatum: Analysis by the Monoclonal Antibody against a Micronuclear Antigen

I obtained the monoclonal antibody 93A against a micronuclear antigen of the ciliate Paramecium caudatum . Immunocytochemical observations showed that the antigen 93A appeared in some portion of the micronucleus in every stage of life cycle. In dividing micronuclei, the antigen appeared mainly in their both poles and in fibrous structures between the poles. These results suggest that the micronuclear antigen 93A may be a component of microtubule organizing center and spindles. During nuclear differentiation in P. caudatum , four among eight postzygotic micronuclei differentiate new macronuclear anlagen and one becomes a new micronucleus and the remaining three degenerate. The micronuclear antigen 93A appeared in all of the eight nuclei in the early stage of macronuclear differentiation but then disappeared in the four macronuclear anlagen and eventually persisted only in the new micronucleus, showing that the newly developing macronuclear anlagen lose the micronuclear antigen 93A during their differentiation.     

冠突伪尾柱虫(Pseudourostyla cristata)接合生殖的研究 Ⅰ.核器演化

在25℃条件下,冠突伪尾柱虫接合生殖全程历时10天左右。接合生殖过程中的核器演化包括:①数十枚老的大核逐步瓦解。电镜观察表明,老的大核是以一种类似于食物泡消化的方式被吸收的,并在此过程中伴有大量溶酶体出现。②仅8枚左右小核中的一枚参与新核器的发生。首先,位于胞口后部的一枚小核膨大并进行一次预备分裂,接着发生三次成熟分裂。每一接合体内形成一枚雄原核和一枚雌原核。雄原核互向对方迁移并与其雌原核融合成为合子核。合子核分裂两次,四枚子核之一发育为大核原基,另一枚发育为小核原基,其余两枚退化。预备分裂和前两次成熟分裂各自产生的两枚子核中,仅一枚进入下一次分裂,另一枚解体消失。在第一次成熟分裂前期,“降落伞”的形成和发展经历着复杂的结构变化,持续一小时以上。③大核原基经过长时间的发育,伴有多线染色体的形成和解体等一系列变化,方达成熟状态。成熟的大核原基以伸长断裂、分叉断裂和哑铃形缢缩三种方式进行分裂,小核原基亦随之分裂,逐步形成具60枚左右大核、8枚左右小核的正常营养体。其后,大核融合,开始配后第一次无性分裂。值得注意的是,大核原基发育到将成熟时,最初的迹象是染色质向大核原基中央集结成团,染色质团与核膜之间充满着匀质的核液。当中央染色质团伸长时,又将