THE TIMING OF MEIOSIS AND DNA SYNTHESIS DURING EARLY OOGENESIS IN THE TOAD, XENOPUS LAEVIS

Recently metamorphosed female Xenopus laevis toads were injected with tritiated thymidine. Animals were kept at 20°C and were sacrificed 1–23 days after isotope injection. Radio-autographs of squash preparations of the ovaries were made. The progress of labeled germ cell nuclei was followed to obtain information on the time course of early meiosis and extra-chromosomal DNA synthesis. Premeiotic S was estimated to take not more than 7 days. Leptotene takes 4 days, zygotene takes 5 days, and pachytene was estimated to be completed in about 18 days. The major period of amplification of the extrachromosomal DNA occurs in pachytene and takes about 13 days. A low level of synthesis was observed before and after this period, in zygotene and late pachytene-early diplotene, extending the total time for extrachromosomal DNA synthesis during meiosis to about 18 days. These data allowed the calculation to be made that one round of replication of the amplified DNA takes between 1.2 and 3.0 days. It was also found that in both oogonial and premeiotic interphases, the nucleolus-associated DNA shows asynchronous (probably late) labeling with respect to the chromosomes.

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An Ultrastructural and Radioautographic Study of Early Oogenesis in the Toad Xenopus Laevis

Journal of Cell Science ◽

10.1242/jcs.12.1.71 ◽

1973 ◽

Vol 12 (1) ◽

pp. 71-93

Author(s):

LESLEY WATSON COGGINS

Keyword(s):

Electron Microscope ◽

Xenopus Laevis ◽

Thymidine Incorporation ◽

Ultrastructural Level ◽

Extrachromosomal Dna ◽

Late Pachytene ◽

Synaptonemal Complexes ◽

Round Nucleus ◽

A Cell ◽

Major Period

Early oogenesis in the toad Xenopus laevis has been investigated at the ultrastructural level, with particular reference to the formation of extrachromosomal DNA. Thymidine incorporation was localized by electron microscope radioautography. In oogonia, the nucleus is irregular in outline and may contain several nucleoli. Oocytes, from premeiotic interphase to late pachytene, are found in cell nests which are estimated to consist of about 16 cells each. Adjacent oocytes within a nest are connected by intercellular bridges and develop synchronously. Each premeiotic interphase-leptotene oocyte has a round nucleus which contains one or two centrally located, spherical nucleoli. Electron-microscope radioautography showed that all nuclei in a cell nest incorporate thymidine synchronously during premeiotic S-phase. In zygotene oocytes, axial cores and synaptonemal complexes are observed in the nucleus and abut against the inner nuclear membrane in the region nearest the centre of the cell nest. The nucleolus is still more-or-less round in outline, but is asymmetrically positioned in the nucleus. It lies near the nuclear envelope on the side of the nucleus furthest away from the attachment of the chromosome ends, that is, nearest the outside of the cell nest. Each nucleolus is surrounded by a fibrillar ‘halo’ of nucleolus-associated chromatin into which a low level of thymidine incorporation occurs during zygotene. This is thought to represent the start of the major period of amplification of the ribosomal DNA. Pachytene is characterized by the presence of synaptonemal complexes in the nucleus. The nucleolus becomes very irregular in outline. The fibrillar area around it, which represents the extrachromosomal DNA, increases in size and thymidine is incorporated over the whole of this region. In late pachytene, many small fibrogranular bodies, the multiple nucleoli, are formed in it. The members of a cell nest become separated from one another at this time and begin to develop asynchronously. In diplotene, synaptonemal complexes are no longer observed in the nucleus. The most prominent structures in the nucleus are now the multiple nucleoli, which increase greatly in number in early diplotene. A large increase in cytoplasmic volume occurs and the oocyte grows in size.

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The development of the tectum in Xenopus laevis: an autoradiographic study

Development ◽

10.1242/dev.28.1.87 ◽

1972 ◽

Vol 28 (1) ◽

pp. 87-115

Author(s):

K. Straznicky ◽

R. M. Gaze

Keyword(s):

Xenopus Laevis ◽

Dna Synthesis ◽

Optic Tectum ◽

Tritiated Thymidine ◽

Dorsal Surface ◽

Autoradiographic Study ◽

Cell Type ◽

Pole Cells ◽

Serial Addition ◽

The Times

The development of the optic tectum in Xenopus laevis has been studied by the use of autoradiography with tritiated thymidine. The first part of the adult tectum to form is the rostroventral pole; cells in this position undergo their final DNA synthesis between stages 35 and 45 or shortly thereafter. Next, the cells comprising the ventrolateral border of the tectum form. These cells undergo their final DNA synthesis at or shortly after stage 45. Finally the cells comprising the dorsal surface of the adult tectum form, mainly between stages 50–55. This part of the tectum originates from the serial addition of strips of cells medially, which displace the pre-existing tissue laterally and rostrally. The formation of the tectum is virtually complete by stage 58. The tectum in Xenopus thus forms in topographical order from rostroventral to caudo-medial. The distribution of labelled cells, several stages after the time of injection of isotope, indicates that, at any one time, a segment of tectum is forming which runs normal to the tectal surface and includes all layers from the ventricular layer out to the surface. In Xenopus, therefore, the times of origin of tectal cells appear to be related not to cell type or tectal layer but to the topographical position of the cells across the surface of the tectum.

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SYNCHRONIZATION OF MITOCHONDRIAL DNA SYNTHESIS IN CHINESE HAMSTER CELLS (LINE CHO) DEPRIVED OF ISOLEUCINE

The Journal of Cell Biology ◽

10.1083/jcb.58.2.340 ◽

1973 ◽

Vol 58 (2) ◽

pp. 340-345 ◽

Cited By ~ 14

Author(s):

Kenneth D. Ley ◽

Marilyn M. Murphy

Keyword(s):

Cell Cycle ◽

Mitochondrial Dna ◽

Dna Synthesis ◽

Cell Cycle Progression ◽

Time Course ◽

Nuclear Dna ◽

Tritiated Thymidine ◽

Chinese Hamster ◽

Chinese Hamster Cells ◽

In Cells

Mitochondrial DNA (mit-DNA) synthesis was compared in suspension cultures of Chinese hamster cells (line CHO) whose cell cycle events had been synchronized by isoleucine deprivation or mitotic selection. At hourly intervals during cell cycle progression, synchronized cells were exposed to tritiated thymidine ([3H]TdR), homogenized, and nuclei and mitochondria isolated by differential centrifugation. Mit-DNA and nuclear DNA were isolated and incorporation of radioisotope measured as counts per minute ([3H]TdR) per microgram DNA. Mit-DNA synthesis in cells synchronized by mitotic selection began after 4 h and continued for approximately 9 h. This time-course pattern resembled that of nuclear DNA synthesis. In contrast, mit-DNA synthesis in cells synchronized by isoleucine deprivation did not begin until 9–12 h after addition of isoleucine and virtually all [3H]TdR was incorporated during a 3-h interval. We have concluded from these results that mit-DNA synthesis is inhibited in CHO cells which are arrested in G1 because of isoleucine deprivation and that addition of isoleucine stimulates synchronous synthesis of mit-DNA. We believe this method of synchronizing mit-DNA synthesis may be of value in studies of factors which regulate synthesis of mit-DNA.

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AUTORADIOGRAPHIC STUDY OF DNA SYNTHESIS AND THE CELL CYCLE IN SPERMATOGONIA AND SPERMATOCYTES OF MOUSE TESTIS USING TRITIATED THYMIDINE

The Journal of Cell Biology ◽

10.1083/jcb.14.1.1 ◽

1962 ◽

Vol 14 (1) ◽

pp. 1-18 ◽

Cited By ~ 246

Author(s):

Valerio Monesi

Keyword(s):

Dna Synthesis ◽

Time Course ◽

Average Duration ◽

Tritiated Thymidine ◽

Periodic Acid ◽

Autoradiographic Study ◽

Type B ◽

Average Life ◽

Periodic Acid Schiff ◽

Average Life Span

Mice were injected intraperitoneally with 15 µc of H3-thymidine. The time course of the labeling in spermatogonia and spermatocytes was studied by using autoradiography on 5 µ sections stained by the periodic acid-Schiff method and hematoxylin over a period of 57 hours after injection. Four generations of type A (called AI, AII, AIII, and AIV), one of intermediate, and one of type B spermatogonia occur in one cycle of the seminiferous epithelium. The average life span is about the same in all spermatogonia, i.e., about 27 to 30.5 hours. The average pre-DNA synthetic time, including the mitotic stages from metaphase through telophase and the portion of interphase preceding DNA synthesis, is also not very different, ranging between 7.5 and 10.5 hours. A remarkable difference exists, however, in the duration of DNA synthesis and of the post-DNA synthetic period. The average DNA synthetic time is very long and is highly variable in type B (14.5 hours), a little shorter and less variable in intermediate (12.5 hours) and AIV (13 hours) spermatogonia, and much shorter and very constant in AIII (8 hours), AII and AI (7 to 7.5 hours) spermatogonia. Conversely, the average post-DNA synthetic time, corresponding essentially to the duration of the prophase, is short and very constant in type B (4.5 hours), longer and variable in intermediate (6 hours) and AIV (8 hours) spermatogonia, and much longer and much more variable in AIII (11 hours), AII and AI (14 hours) spermatogonia. The premeiotic synthesis of DNA takes place in primary spermatocytes during the resting phase and terminates just before the visible onset of the meiotic prophase. Its average duration is 14 hours. No further synthesis of DNA takes place in later stages of spermatogenesis.

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Initiation of DNA synthesis by kinetin and experimental factors in tobacco pith tissues in vitro

Canadian Journal of Botany ◽

10.1139/b71-217 ◽

1971 ◽

Vol 49 (9) ◽

pp. 1541-1549 ◽

Cited By ~ 21

Author(s):

Antoine Simard

Keyword(s):

Dna Synthesis ◽

Liquid Scintillation ◽

Nuclear Dna ◽

Tritiated Thymidine ◽

Scintillation Counting ◽

Basic Medium ◽

Control Medium ◽

Cell Nuclei ◽

Counting Methods

Since previous investigations had suggested that kinetin, like auxin, may initiate DNA synthesis in tobacco pith cells, a study was undertaken to learn whether the observed incorporation of tritiated thymidine into nuclear DNA in the presence of kinetin could be explained by different experimental factors.Pith tissues were isolated and allowed to rest a few days after excision and they were then placed on White's basic medium or on that medium supplemented with either one or both of the growth regulators in the presence of tritiated thymidine.The results of those studies, obtained by radioautographic and liquid scintillation counting methods, showed no statistically significant differences between pith tissues kept on the control medium and those on kinetin-containing medium. Similar tissues placed on an auxin or auxin- and kinetin-containing medium showed, as expected, a significant incorporation of tritiated thymidine into the DNA of pith cell nuclei.

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Review Lecture - Replication of DNA in the chromosomes of eukaryotes

Proceedings of the Royal Society of London Series B Biological Sciences ◽

10.1098/rspb.1972.0039 ◽

1972 ◽

Vol 181 (1062) ◽

pp. 19-41 ◽

Cited By ~ 139

Keyword(s):

Dna Synthesis ◽

Double Helix ◽

Tritiated Thymidine ◽

Somatic Cells ◽

S Phase ◽

Lampbrush Chromosomes ◽

Chromosomal Dna ◽

Replication Process ◽

Initiation Point ◽

Dna Double Helix

The evidence that each chromatid of a eukaryotic organism contains only one DNA double helix comes from a variety of observations. It begins with the autoradiographic demonstration by J. H. Taylor that tritiated thymidine, incorporated into chromosomes during one round of DNA synthesis, is present in both chromatids at the first division after labelling, but in only one chromatid after a further round of DNA synthesis accomplished in the absence of label. Further evidence comes from those experiments which demonstrate that when two sister chromatids break and fuse one with the other, each chromatid behaves as though it contained two chains of opposite polarity, fusion between chains being restricted to those of like polarity. J. G. Gall’s study of the kinetics of digestion of lampbrush chromosomes by pancreatic DNase also supports the view that each chromatid contains only two polynucleotide chains which are cleaved by this enzyme independently of one another; while O. L. Miller’s observations on the dimensions of the fibres remaining after lampbrush chromosomes have been digested by trypsin only allow for there being two polynucleotide chains per chromatid. By means of the technique of DNA fibre autoradiography devised by J. A. Huberman and A. D. Riggs, the units involved in replicating the chromosomal DNA of somatic cells of Xenopus have been compared with those of Triturus . Both these organisms have initiation points for DNA replication that are arranged in tandem, and from each initiation point replication proceeds in opposite directions at divergent forks. The intervals between initiation points in Xenopus range from about 20 to 125 µ m apart, whereas those of Triturus are much more widely separated. At 25 °C replication of DNA in Xenopus somatic cells proceeds at 9 µ m per hour one-way at each fork, whereas the corresponding rate in Triturus is 20 µ m per hour. Triturus somatic cells take about 4 times longer than comparable cells of Xenopus to replicate their DNA. The Triturus genome contains about 10 times as much DNA as the Xenopus genome, and comparison of the replication process in these two organisms indirectly adds weight to the view that the Triturus genome is 10 times longer than that of Xenopus , rather than that it contains 10 times as many DNA double helices per chromatid. DNA fibre autoradiography has also been used to study replication in Triturus spermato-cytes. The round of DNA synthesis just before meiosis in Triturus is an exceptionally long-drawn-out process, taking 9 to 10 days for completion at 16 °C. This lengthy S-phase is not occasioned by abnormally slow replication, the rate being 12 µ m per hour one-way at 18 °C, nor is it the result of an exceptional staggering of replication starts. Instead it appears to be correlated with a gross reduction in the number of initiation points for replication. i.e. with an increase in the lengths of the replicating units. A rough calculation suggests that each meiotic chromomere may correspond to a unit of replication during the pre-meiotic S-phase.

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Preliminary Evaluation of a Weibull Function for Fitting Slow-Component Eye Velocity over the Time Course of Caloric-Induced Nystagmus

Journal of Speech Language and Hearing Research ◽

10.1044/jshr.3203.681 ◽

1989 ◽

Vol 32 (3) ◽

pp. 681-687 ◽

Cited By ~ 1

Author(s):

C. Formby ◽

B. Albritton ◽

I. M. Rivera

Keyword(s):

Time Course ◽

Slow Component ◽

Weibull Function ◽

Preliminary Evaluation ◽

Mathematical Function ◽

Before And After ◽

Best Fitting ◽

Eye Velocity ◽

Fitting Parameters ◽

Weibull Equation

We describe preliminary attempts to fit a mathematical function to the slow-component eye velocity (SCV) over the time course of caloric-induced nystagmus. Initially, we consider a Weibull equation with three parameters. These parameters are estimated by a least-squares procedure to fit digitized SCV data. We present examples of SCV data and fitted curves to show how adjustments in the parameters of the model affect the fitted curve. The best fitting parameters are presented for curves fit to 120 warm caloric responses. The fitting parameters and the efficacy of the fitted curves are compared before and after the SCV data were smoothed to reduce response variability. We also consider a more flexible four-parameter Weibull equation that, for 98% of the smoothed caloric responses, yields fits that describe the data more precisely than a line through the mean. Finally, we consider advantages and problems in fitting the Weibull function to caloric data.

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Microfluidic extraction and stretching of chromosomal DNA from single cell nuclei for DNA fluorescence in situ hybridization

Biomedical Microdevices ◽

10.1007/s10544-011-9621-8 ◽

2012 ◽

Vol 14 (3) ◽

pp. 443-451 ◽

Cited By ~ 10

Author(s):

Xiaozhu Wang ◽

Shin-ichiro Takebayashi ◽

Evans Bernardin ◽

David M. Gilbert ◽

Ravindran Chella ◽

...

Keyword(s):

In Situ Hybridization ◽

Fluorescence In Situ Hybridization ◽

Single Cell ◽

Cell Nuclei ◽

Chromosomal Dna

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TGF-β1 increases permeability of ciliated airway epithelia via redistribution of claudin 3 from tight junction into cell nuclei

Pflügers Archiv - European Journal of Physiology ◽

10.1007/s00424-020-02501-2 ◽

2021 ◽

Author(s):

Carolin Schilpp ◽

Robin Lochbaum ◽

Peter Braubach ◽

Danny Jonigk ◽

Manfred Frick ◽

...

Keyword(s):

Time Course ◽

Atopic Asthma ◽

Ciliated Cells ◽

Cell Nuclei ◽

Tissue Remodelling ◽

Similar Time ◽

Airway Epithelia ◽

Epithelial Integrity ◽

Motile Cilia ◽

Tgf Β1

AbstractTGF-β1 is a major mediator of airway tissue remodelling during atopic asthma and affects tight junctions (TJs) of airway epithelia. However, its impact on TJs of ciliated epithelia is sparsely investigated. Herein we elaborated effects of TGF-β1 on TJs of primary human bronchial epithelial cells. We demonstrate that TGF-β1 activates TGF-β1 receptors TGFBR1 and TGFBR2 resulting in ALK5-mediated phosphorylation of SMAD2. We observed that TGFBR1 and -R2 localize specifically on motile cilia. TGF-β1 activated accumulation of phosphorylated SMAD2 (pSMAD2-C) at centrioles of motile cilia and at cell nuclei. This triggered an increase in paracellular permeability via cellular redistribution of claudin 3 (CLDN3) from TJs into cell nuclei followed by disruption of epithelial integrity and formation of epithelial lesions. Only ciliated cells express TGF-β1 receptors; however, nuclear accumulations of pSMAD2-C and CLDN3 redistribution were observed with similar time course in ciliated and non-ciliated cells. In summary, we demonstrate a role of motile cilia in TGF-β1 sensing and showed that TGF-β1 disturbs TJ permeability of conductive airway epithelia by redistributing CLDN3 from TJs into cell nuclei. We conclude that the observed effects contribute to loss of epithelial integrity during atopic asthma.

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