Nuclear DNA synthesis during the induction of embryogenesis in cultured microspores and pollen of Brassica napus L.

The dynamics of nuclear DNA synthesis were analysed in isolated microspores and pollen of Brassica napus that were induced to form embryos. DNA synthesis was visualized by the immunocytochemical labelling of incorporated Bromodeoxyuridine (BrdU), applied continuously or as a pulse during the first 24 h of culture under embryogenic (32 °C) and non-embryogenic (18 °C) conditions. Total DNA content of the nuclei was determined by microspectrophotometry. At the moment of isolation, microspore nuclei and nuclei of generative cells were at the G1, S or G2 phase. Vegetative nuclei of pollen were always in G1 at the onset of culture. When microspores were cultured at 18 °C, they followed the normal gametophytic development; when cultured at 32 °C, they divided symmetrically and became embryogenic or continued gametophytic development. Because the two nuclei of the symmetrically divided microspores were either both labelled with BrdU or not labelled at all, we concluded that microspores are inducible to form embryos from the G1 until the G2 phase. When bicellular pollen were cultured at 18 °C, they exhibited labelling exclusively in generative nuclei. This is comparable to the gametophytic development that occurs in vivo. Early bicellular pollen cultured at 32 °C, however, also exhibited replication in vegetative nuclei. The majority of vegetative nuclei re-entered the cell cycle after 12 h of culture. Replication in the vegetative cells preceded division of the vegetative cell, a prerequisite for pollen-derived embryogenesis.     

A cytophotometric analysis of the structure of hypothalamic cell populations in the frog,Rana temporaria (L.), with special reference to seasonal changes in chromatin status

Summary We used cytophotometry after the Feulgen reaction and UV cytophotometry to measure the DNA content of quiescent cells of the hypothalamic preoptic region (HPR) of adult and juvenile frogs (Rana temporaria) that had been caught in their natural habitat in winter, spring and summer. The histone-to-DNA ratio in cell nuclei was cytophotometrically determined using a combined Feulgen, heparine and alcian-blue staining procedure. The vast majority of HPR cells studied had nuclei with a diploid DNA content. However, we observed great variability in the Feulgen-DNA content of the HPR cell population, which was not detected in the diploid standard (hepatocytes). This heterogeneity in the diploid sample of the HPR cell populations was always greater in prespawning frogs and may have been due to differences in the chromatin arrangement in nuclei. About 1% of cells had a DNA content either ranging between diploid and tetraploid levels (H2C cells) or at the tetraploid level (4C and 2C x 2 cells). The proportion of these cells was not affected by the age of the animals or the annual cycle, thus suggesting that there is no age-related increase in the mean DNA content in the frog HPR. The mean DNA contents of H2C and 4C cells were much higher than those in the standard (hepatocytes). This cannot be simply attributed to the presence of different amounts of nuclear proteins, but rather indicates that at least a certain proportion of the highest DNA contents may be due to a real extra-DNA synthesis.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

Patterns of endopolyploidy and 2C nuclear DNA content (Feulgen) inScilla (Liliaceae)

The Feulgen-DNA content of 3558 nuclei from 21 different tissues and organs ofScilla decidua (Liliaceae) was measured by a scanning cytophotometer interfaced to a computer. The basic nuclear DNA content (2 C value) was 13.62 pg, and 71 per cent of the nuclei were polyploid. The highest DNA values were found in the antipodal cells of the ovule, and the elaiosomes of the seeds (512 C). In addition to polyploidy, the 2 C values exhibited tissue-specific variation which was statistically significant (0.05% level of probability), It is suggested that differential DNA replication and endopolyploidization may be basic factors in the complex mechanism of cell and tissue differentiation.Dedicated to Professor Dr.Lothar Geitler in honour of his 80th birthday.     

Quantitation of cell kinetic responses using simultaneous flow cytometric measurements of DNA and nuclear protein

A rapid procedure was developed for the simultaneous flow cytometric analysis of nuclear protein using fluorescein isothiocyanate, and DNA using propidium iodide in isolated nuclei. The staining procedure did not involve centrifugation and was easily adapted to the staining of human peripheral blood lymphocytes stimulated with phytohemagglutinin, EL4 murine lymphoid tumor cells in suspension culture, and R3327-G rat prostatic adenocarcinoma solid tumor specimens. Histograms of unstimulated and PHA-stimulated HPBL perturbed by actinomycin D, hydroxyurea, 3H-TdR, colcemid, or hydroxyurea + colcemid showed that 1) resting, noncycling G1 (G1Q) cells are distinguished from late G1 (G1AB) cells, 2) early G2 (G2A) cells are distinguished from late G2 (G2B) cells, and 3) mitotic cells are distinguished from G2 cells. Treatment with hydroxyurea resulted in a build-up of cells having high nuclear protein content and 2C DNA content (G1AB), while incubation with 3H-TdR caused an increase in the number of cells with high nuclear protein content and 4C DNA content (G2B). Colcemid-blocked mitotic cells were identified as having low nuclear protein content (lower than G2A nuclei) and 4C DNA content. The nuclear DNA/protein histograms of untreated and colcemid-treated log-phase EL4 cells provided information concerning G1A, G1B, S, G2A, G2B, and M. The method was also used to quantitate the response of androgen-sensitive rat prostatic R3327-G tumors to androgen deprivation following castration. Sample preparation and staining for correlated nuclear DNA/protein measurements takes approximately the same amount of time as for single parameter nuclear DNA measurements.     

SOME OBSERVATIONS ON DNA CONTENT AND CELL AND NUCLEAR VOLUME GROWTH IN THE DEVELOPING XYLEM CELLS OF CERTAIN HIGHER PLANTS

List , Albert , Jr . (Douglass Coll., Rutgers U., New Brunswick, N. J.) Some observations on DNA content and cell and nuclear volume growth in the developing xylem cells of certain higher plants. Amer. Jour. Bot. 50(4): 320–329. Illus. 1963.—The developing metaxylem cells of Acorus calamus roots undergo an over-all growth in cell and nuclear volume that may be expressed roughly as a constant ratio of relative growth rates. Within this over-all growth picture, however, there is a periodicity of both nuclear and cell volume growth. Other plants such as Peltandra, Eleocharis, and Dennstaedtia undergo a similar volume growth. Marsilea tracheary elements have an increase in nuclear number per cell by simultaneous divisions. Arisaema metaxylem nuclei go through a series of DNA doublings correlated with nuclear volume doubling and cell volume increase, at least up to the 16- or 32-ploid level. The cells display some tendency to fall into size classes, expressing a pulsation in growth. A fluctuating alternation or stepwise growth of cell and nucleus appears to describe the data more suitably than the allometric growth equation. In Zea, the cell and nuclear volumes for metaxylem cells fit a fluctuating envelope better than the straight line, and there is again the probability that cell volumes fall into size classes related to nuclear volume class. The DNA content of the nuclei was determined to fall into a frequency distribution having peaks at the 4-, 8-, 16-, and 32-ploid equivalent, agreeing with an earlier report for diploid maize. DNA content was highly correlated with nuclear volume in the over-all growth of the metaxylem cells.     

Change with age of feulgen-DNA values in the blood-sucking insect,Triatoma infestans klug

Summary Feulgen-DNA values were evaluated cytophotometrically in the Malpighian tubes of T. infestans at the nymphal, adult and ageing life periods. In all cases the total nuclear Feulgen-DNA contents were found to be distributed within the 32C and 64C classes. However, part of the Feulgen-DNA values is shifted to lower intervals in ageing insects, as compared with nymphal and adult individuals, promoted by decrease in Feulgen-DNA values of the euchromatins. Changed response to Feulgen reaction due to some aging-induced alteration in DNP complexes, and/or breaks and loss of DNA in the euchromatins, as part of the senescence process, are ascribed to be responsible for such phenomenon.     

DNA-Feulgen cytophotometric determination of genome size for the freshwater-invading copepod Eurytemora affinis.

Variation in nuclear DNA content within some eukaryotic species is well documented, but causes and consequences of such variation remain unclear. Here we report genome size of an estuarine and salt-marsh calanoid copepod, Eurytemora affinis, which has recently invaded inland freshwater habitats independently and repeatedly in North America, Europe, and Asia. Adults and embryos of E. affinis from the St. Lawrence River drainage were examined for somatic cell DNA content and the presence or absence of embryonic chromatin diminution, using Feulgen-DNA cytophotometry to determine a diploid or 2C genome size of 0.6-0.7 pg DNA/cell. The majority of somatic cell nuclei, however, have twice this DNA content (1.3 pg/nucleus) in all of the adults examined and possibly represent a population of cells arrested at the G2 stage of the cell cycle or associated with some degree of endopolyploidy. Both suggestions contradict assumptions that DNA replication does not occur in adult tissues during the determinate growth characteristic of copepods. Absence of germ cell nuclei with markedly elevated DNA values, commonly found for species of cyclopoid copepods that show chromatin diminution, indicates that E. affinis lacks this trait. The small genome size and presumed absence of chromatin diminution increase the potential utility of E. affinis as a model for genomic studies on mechanisms of adaptation during freshwater invasions.     

Microdensitometric and autoradiographic comparison of the DNA contents of foetal and adult rat liver nuclei

Summary Glare-corrected, scanning Feulgen microdensitometry and ³H-thymidine autoradiography were applied to squash preparations of rat 18-day foetal and maternal liver cells, and to smears of maternal blood. No significant differences were found between the mean Feulgen-DNA contents of autoradiographically unlabelled diploid foetal and maternal hepatocytes. The Feulgen-DNA contents of other unlabelled foetal and maternal hepatocytes were also as predicted by the DNA-constancy hypothesis, i.e. were twice or four times that of diploid cells. Small (less than about 4%) but statistically significant discrepancies in the mean Feulgen-DNA contents of foetal haemopoietic cells and adult leucocytes were attributable to uncorrected residual distribution and chromatic errors in the microdensitometry.None of the 371 maternal nuclei measured had Feulgen-DNA contents substantially (i.e. more than ±10%) different from a modal value. About 12% of these nuclei were classified as labelled. Evidence was found suggesting a significantly non-random distribution of background grains in the autoradiographs, which would materially affect the proportion of cells incorrectly classified. After taking this factor into account there seems no reason to suppose that the apparently labelled adult nuclei were in fact synthesising DNA.Of 376 foetal cells measured, 107 had inter-modal Feulgen-DNA contents. Eleven of these were classified as unlabelled. All the inter-modal cells were however probably in the S-phase of the cell cycle, statistical variation in autoradiographic grain distribution accounting for those appearing to be unlabelled.Our results are consistent with the DNA-constancy hypothesis, and are at variance with previous claims for the existence of metabolic DNA in adult and/or foetal rat hepatocytes.     

Endopolyploidy in follicle epithelial cells around the nurse chamber during oogenesis in Chrysomya putoria (Diptera,Calliphoridae)

Feulgen-DNA values and nuclear areas were evaluated microspectrophotometrically for epithelial cells of the ovarian follicle during oogenesis in Chrysomya putoria. The aim was to investigate whether polyploidization occurred in the cells surrounding the nurse chamber and/or in those around the oocyte as well as whether different DNA amounts were found regarding the cell types considered. Four DNA endoreduplicative cycles could be demonstrated for the epithelial cells regardless of their localization on the follicle, during oogenesis. A small percentage of epithelial cells reaches a 32C degree. The nuclear area, however, did not increase at the same rate in cells covering the oocyte as in those covering the nurse chamber, in some of the oogenesis stages. The meaning of endopolyploidy for these cells is discussed, considering reports on relevance of secretory activities and their maintenance in polyploid cell systems.     

Change with age of Feulgen-DNA values in the blood-sucking insect, Triatoma infestans Klug.

Feulgen-DNA values were evaluated cytophotometrically in the Malpighian tubes of T. infestans at the nymphal, adult and ageing life periods. In all cases the total nuclear Feulgen-DNA contents were found to be distributed within the 32C and 64C classes. However, part of the Feulgen-DNA values is shifted to lower intervals in ageing insects, as compared with nymphal and adult individuals, promoted by decrease in Feulgen-DNA values of the euchromatins. Changed response to Feulgen reaction due to some aging-induced alteration in DNP complexes, and/or breaks and loss of DNA in the euchromatins, as part of the senescence process, are ascribed to be responsible for such phenomenon.     

Initiation of DNA replication in nuclei from quiescent cells requires permeabilization of the nuclear membrane

We have investigated the replication capacity of intact nuclei from quiescent cells using Xenopus egg extract. Nuclei, with intact nuclear membranes, were isolated from both exponentially growing and contact- inhibited BALB/c 3T3 fibroblasts by treatment of the cells with streptolysin-O. Flow cytometry showed that > 90% of all contact- inhibited cells and approximately 50% of the exponential cells were in G0/G1-phase at the time of nuclear isolation. Intact nuclei were assayed for replication in the extract by incorporation of [alpha- 32P]dATP or biotin-dUTP into nascent DNA. Most nuclei from exponential cells replicated in the egg extract, consistent with previous results showing that intact G1 nuclei from HeLa cells replicate in this system. In contrast, few nuclei from quiescent cells replicated in parallel incubations. However, when the nuclear membranes of these intact quiescent nuclei were permeabilized with lysophosphatidylcholine prior to addition to the extract, nearly all the nuclei replicated under complete cell cycle control in a subsequent incubation. The ability of LPC-treated quiescent nuclei to undergo DNA replication was reversed by resealing permeable nuclear membranes with Xenopus egg membranes prior to extract incubation demonstrating that the effect of LPC treatment is at the level of the nuclear membrane. These results indicate that nuclei from G1-phase cells lose their capacity to initiate DNA replication following density-dependent growth arrest and suggest that changes in nuclear membrane permeability may be required for the initiation of replication upon re-entry of the quiescent cell into the cell cycle.     

Nuclear DNA content and the control of chloroplast replication in wheat leaves

During development of the first leaf of breadwheat (Triticum aestivum L.) the number of chloroplasts per mesophyll cell increases between three- and four-fold. To establish if chloroplast replication is accompanied by endoreduplication, the nuclear DNA content of the cells was determined by chemical assay of isolated nuclei from mesophyll protoplasts and by microdensitometry of nuclei in mesophyll tissue. The DNA content of the nuclei was constant (27 to 32 pg) at each phase of chloroplast replication. Approximately 93% of the cells had a nuclear DNA content close to the 2C value of 32 pg. It is concluded that chloroplast replication is not dependent on nuclear endoreduplication in seedling leaves of wheat.     

Image analysis of Feulgen-stained c-H-ras-transformed NIH/3T3 cells

Feulgen-DNA content, nuclear phenotypes, and levels of chromatin condensation were evaluated by image analysis in NIH/3T3 cells transformed with the c-H-ras oncogene of T24 cells. Three nuclear phenotypes, differing from those of untransformed control cells and defined in terms of patterns of chromatin condensation, were demonstrated microspectrophotometrically for the tumor cells. Polyploidy could only be observed in nuclei with extensive and deeply stained areas covered with condensed chromatin, i.e., only in a small fraction of the tumor cell nuclear population. The increased chromatin condensation that appeared with cell transformation affected the euchromatin zones. The image analysis provided data that, compared with those obtained in other situations involving cell transformation, could be relevant to the understanding of changes in chromatin supraorganization related to tumorigenesis and to tumor cell diagnosis.     

Cytofluorometry on cells isolated from paraffin sections after blocking of the background fluorescence by azocarmin G

Summary A technique for isolation of cells from paraffin embedded tissue is indispensable for the performance of Feulgen-DNA cytofluorometry in parallel with the definition of histological characteristics. Background fluorescence due to nonspecific dye-binding by a pseudo-plasmal reaction is usually found to be so intense on cells isolated from formalin-fixed tissues, that we are often forced to abandon quantitative DNA determinations. In the present work, we report that fixation of tissues with Carnoy's fixative for 12 h at 5° C not only reduces nonspecific dye-binding but also facilitates the process of cell isolation. Furthermore, we find that pre-treatment of cells isolated from Carnoy-fixed tissues with acidic azocarmin G solution completely blocks nonspecific dye-binding in subsequent acriflavine Feulgen nuclear staining. This combination of techniques for specimen preparation enables us to carry out Feulgen-DNA cytofluorometry on cells isolated from histological sections with satisfactorily low coefficients of variation (less than 8%). The techniques should be widely applicable for parallel DNA determinations and histology.     

Nuclear RNA quantification in protoplast cell-cycle phases

Using acridine orange staining and flow cytometry the DNA and RNA levels (arbitrary units) of individual cells may be established. Here, this method has been applied to nuclei isolated from plant protoplasts during culture. The specificity of the technique has been validated for such plant material; ribonuclease markedly reduced nuclear staining without modifying the DNA histogram; ribonuclease inhibitor prevented the action of released cell nucleases; and protoplasts cultivated with actinomycin D did not synthesize RNA. First RNA synthesis was evident 18 h after Petunia hybrida protoplasts had been put into culture. An increase of RNA above a critical level was required for cells to be able to initiate DNA replication from G1, termed G1B. G2 nuclei had an RNA:DNA ratio similar to that of G1 nuclei.     

Image analysis of rat satellite cell proliferation in vitro

Myogenic cells were isolated from adult rat skeletal muscles and cultured in vitro. Cell proliferation was analyzed between days 1 and 14. The cell cycle phases were determined by examining Feulgen-stained cultures with a cell image processor. The nuclei were automatically analyzed by calculating 18 parameters relating to the texture and densitometry of chromatin and the shape of each nucleus. Cell cycle phases were characterized (Moustafa and Brugal, 1984). The recognition methods made it possible to analyse the nuclei of the myogenic cell populations which were either involved in each phase of the mitotic cycle, or left out of the cycle after fusion into myotubes.After 3 hr of culture 10% of the cell population was involved in the cell cycle. In the presence of foetal calf serum, this percentage increased until day 3 after plating. At that time, the DNA content of 28.2% of the cell population was higher than 3C, whereas it is 2C in G1 or G0 nuclei; image analysis showed that 42% of these cells were in S or G2 phase. From day 4, the proliferation rate gradually slowed down until day 8. After day 8, when numerous myotubes differentiated, the percentage of S and G2 phase cells had diminished to between 3 and 8%. The percentage of nuclei in G0 increased when the first myotubes differentiated around day 5. Myotube nuclei were largely in G0. When horse serum was added to the culture medium on day 4 to enhance myotube differentiation, significant cell proliferation was observed before cell fusion.These methods of analysis give the first daily pattern of myogenic cell proliferation and fusion in a cell population isolated from adult muscles.     

Microphotometric Determination of DNA Contents of Early Developmental Pollen Grains in Tobacco Anther Culture

The Feulgen-DNA contents of microspores, vegetative and generative nuclei of tobacco pollen grains in vivo and in anther culture have been determined by microphotometry. 1. The values of DNA content of vegetative and generative nuclei of the pollen grains selected at definite developmental stages vary between 1C and 2C levels, which coincide with the role of the dynamics of DNA in haploid cell cycle. This method applied in the study of androgenesis in anther culture is proved successful and valid. 2. By the cytomorphological investigation on androgenesis, the pollen embryoid in this experiment results from repeated divisions of the vegetative cell within the pollen grains. 3. In mature pollen grains of the same variety of tobacco in vivo, DNA replication has not occured in vegetative nuclei, in which the level of DNA remains in 1C. 4. In the cultured anthers after 8 days innoculation, 30% of the total pollen grains measured indicate that the vegetative nuclei have completed DNA replication and show 2C level. The pollen grains which have the potential to differentiate into the embryogenie pollen grains, may be distinguished from non-embryogenie ones by this method before any cytomorphological sign appears. The significance of this method in the study of the mechanism of androgenesis is discussed.     

DNA synthesis in the second and third cell cycles of mouse preimplantation development. A cytophotometric study

Female Swiss mice were sacrificed at 2 h intervals between 16–30 and 40–56 h after insemination. One-, 2- and 4-cell embryos were stained by the Feulgen method and cytophotometric measurement of their nuclear DNA content was carried out. The cells with 2C and 4C DNA content were assumed to be in G1 and G2 phase and those with intermediate DNA content in S phase of the cell cycle. The fractions of cells which had passed a given phase of the cell cycle were calculated for various times after insemination and utilized for measurements of the second and third cell cycle timing. Results of measurements for the second cell cycle: G1 phase 1.3 h, S phase 6.1 h, G2 phase 15.4 h, whereas for the third cell cycle: G1 phase 1.6 h, S phase 7.4 h, G2 phase 0.5 h. The first cleavage division was calculated as 1.6 h, the second as 1.3 h and the third as 1.2 h. Complete intra-embryonic synchronization of the DNA-synthesizing nuclei was preserved during the entire synthesis phase of 2-cell embryos, while in 4-cell embryos they were slightly asynchronized. Among mitotic cells of the first cleavage division and G1 cells of 2-cell embryos a slight interembryonic asynchronization was found which deepened during subsequent cell cycle phases.