Feulgen Cytophotometry Research Articles

Timing of the phases of the cell cycle with tritiated thymidine and Feulgen cytophotometry during the period of synchronous division in Lymnaea

ABSTRACT The duration of the phases of the cell cycle during the 1–, the 2– and the 4–cell stage of the Lymnaea egg were determined with [3H]thymidine and with Feulgen cytophotometry. The M, S and G2 phases occupy 48, 27 and 25% of the first three cell cycles. A G1 phase cannot be observed. Only from the 4-cell stage was [3H]thymidine readily incorporated into DNA. The theory that an increase in respiration during the S phase of the 4-cell stage is connected with the energy requirements of DNA synthesis is discussed.

A cytophotometric study of benign and malignant phaeochromocytomas.

Measurement of nuclear D.N.A. content of fifteen phaeochromocytomas was made by quantitative Feulgen cytophotometry, using the Vickers M 85 integrating microdensitometer. The tumours fell into two groups. The first and larger group (12 tumours), had a mode corresponding to a diploid (2n) D.N.A. content, and a very wide range of values, with nuclei measuring up to 40n. In contrast the second group (3) had a hyperdiploid or triploid mode and a smaller range of values. Analysis of case records showed that all three tumours in the second group, and none of those in the first group, had metastasised.

Primitive erythropoiesis in early chick embryogenesis. II. Correlation between hemoglobin synthesis and the mitotic history.

Primitive erythroblasts in the circulating blood of the chick embryo continue to divide while synthesizing hemoglobin (Hb). Hb measurements on successive generations of erythroblasts show that there is a progressive increase in the Hb content of both interphase and metaphase cells. Furthermore, for any given embryo the Hb content of metaphase cells is always significantly greater than that of interphase cells. The distribution of Hb values for metaphase cells suggests that there are six Hb classes corresponding to the number of cell cycles in the proliferative phase. The location of erythroblasts in the cell cycle was determined by combining Feulgen cytophotometry with thymidine radioautography on the same cells. Measurements of the Hb content for erythroblasts in different compartments of the cell cycle (G1, S, G2, and M) show a progressive increase through the cycle. Thus, the amount of Hb per cell is a function of the number of cell divisions since the initiation of Hb synthesis and, to a lesser degree, the stage of the cell cycle. Earlier generations of erythroblasts synthesize Hb at a faster rate than the terminal generation. Several models have been proposed to explain these findings.

Histoenzymological and cytophotometric studies on the variations of some enzymatic activities in different cell cycle phases in cultured cells

Cyclic variations of some enzymatic activities are demonstrated in hamster cells in vitro by a combination of histoenzymological techniques, Feulgen cytophotometry and 3H-thymidine radioautography. The periods of activity would be particularly important in the S and G2 phases. The comparison of normal cells and cancer cells does not show significant difference in this pattern of enzymatic activities.

Decreased DNA content of birch (Betula) chromosomes at high ploidy as determined by cytophotometry

Relative amounts of DNA were determined on telophase nuclei by Feulgen cytophotometry for euploid taxa of birch (Betula) with somatic chromosome numbers of 28, 42, 56, 70, and 84. A direct correlation was found between observed DNA absorbance and chromosome number except for plants of B. papyrifera with 84 somatic chromosomes. The DNA density value for nuclei of the 84-chromosome plants fitted a 1∶2.25 ratio instead of the expected 1∶3.0 ratio. The DNA density value for these plants was calculated to be approximately equivalent to plants which would possess 63 somatic chromosomes. The average DNA value per chromosome was 2.73 for the 84-chromosome plants in contrast to 3.50 per chromosome in each of the lower euploids. Nuclear diameters of the 84-chromosome plants were directly related to chromosome number and not to DNA density value. The genomic number of Betula was considered to be x=7, rather than x=14, since a DNA value equivalent to 63 chromosomes is a multiple of 7 and not 14. Diploid birch species (2n=2x=28), therefore, would actually be tetraploids (2n=4x=28). The reduction in DNA content may be an adaptation for the establishment of higher ploidy in birches.

Effect of estrogens and gestagens on the initiation of DNA synthesis in the genital tract of ovariectomized mice

The initiation of DNA synthesis in vaginal and endometrial epithelial cells of estrogen or gestagen treated mice was studied by means of 3H-thymidine autoradiography and quantitative Feulgen cytophotometry. With the doses employed, estrogen primed castrated mice showed no differences in the reaction to both investigated hormones. After a period of 7 h, a stimulation of DNA synthesis begins in vaginal as well as endometrial epithelial cells and increases until 14 h, when it reaches a plateau. The cytophotometrical DNA measurements agree satisfactorily with the autoradiographic results, when the morphological particularities of epithelial architecture are considered.

Feulgen cytophotometry of pine nuclei; effects of fixation, role of formalin.

The effects of 5 fixatives: FAA, Carnoy's, Craf III, formalin and glutaraldehyde were analyzed for use in quantitative Feulgen cytophotometry of pine embryo tissues. Craf III and glutaraldehyde had serious deficiencies because they depressed the absorption peak, severely interfered with DNA extraction and in the case of glutaraldehyde there was considerable cytoplasmic dye-binding. Neutral 10% formalin gave good tissue fixation but did not permit the degree of enzymatic or acid extraction of DNA as did Carnoy's solution. Haupt's adhesive, with the usual 4% formalin as a hardener, at temperatures of 45-56 C completely prevented the enzymatic extraction of nuclear DNA by DNase and also greatly increased the resistance of the DNA to mineral acid hydrolysis. Denaturation of DNA by formalin appeared to be responsible for these results. Absorption was linearly related to both section thickness and DNA concentration per nucleus.

DNA content of cervical intraepithelial neoplasia studied by two-wavelength Feulgen cytophotometry

The DNA content of individual nuclei in biopsies of normal cervical epithelium, dysplasia, and carcinoma in situ was estimated by microspectrophotometric measurements of Feulgen-stained sections. The normal epithelium had a diploid modal value and a diploid-tetraploid range whereas in all cases of dysplasia and carcinoma in situ there were significant numbers of cells with a DNA content significantly above this range and with an abnormal mode. The degree of deviation of the DNA values from the normal distribution was least in the mild dysplasias but became more pronounced in the higher grade lesions. These observations are consistent with the concept that the lesions studied are related as part of a continuum.

Species relationships in the genus Tulipa

The relationships of diploid and polyploid species belonging to the sub-section Eriostemones of the genus Tulipa have been investigated from the point of view of chromosome morphology, relative DNA values, and meiotic pairing properties. Karyotype morphology is basically the same for all species and eight principal chromosome classes can be distinguished. The range of DNA values obtained by Feulgen cytophotometry for the diploid species is relatively small and provides little assistance in ascertaining the pattern of ploidy involved in the evolution of the group. Partial asynapsis and reverse loop pairing in several of the diploids at pachytene reveals a degree of structural differentiation which may be the outcome of hybridity between species. Significantly, these anomalies are rarely encountered at the equivalent tetraploid levels and a scheme is proposed to account for this behaviour.

Cytophotometric studies on the DNA content in diploid and polyploid enchytraeidae (Oligochaeta)

The amount of DNA in nerve nuclei was determined by Feulgen cytophotometry in 45 species and cytotypes. The DNA value of spermatids, spermatogonial nuclei and nerve nuclei in newly hatched worms was determined in some species. The total variation in DNA content ranges from a relative value of 0.40 to 4.85. The DNA content in diploid species ranges from 0.40 to 1.12. In two genera (Lumbricillus and Enchytraeus) wide diversification in chromosome numbers at the diploid level is accompanied by wide variation in DNA values, whereas other genera and species groups (Mesenchytraeus, Henlea and Marionina), which are conservative in this respect, are closer in DNA values. In the genus Fridericia constancy in chromosome number is associated with wide variation in DNA values, this may be due to an increase in DNA taking place in old individuals. In 9 out of 10 cases nearly identical ratios are found between chromosome numbers and DNA values in polyploids and related diploids or lower polyploids. Differential polyteny is recorded in two cases.

Chromosome size and DNA content of species of anemone L. and related genera (Ranunculaceae)

Relative amounts of DNA were determined by Feulgen cytophotometry in 22 diploid species of Ranunculaceae (n=7, 8, 9) representing six genera, and exhibiting large differences in chromosome size, but no marked differences in karyotype pattern. Chemical determination of absolute amounts of DNA for six of these species, allowed conversion of all the photometric data into absolute units of DNA. The mean DNA content per nucleus varied from.13×10−11gm in Aquilegia to 5.25×10−11gm in species of Anemone in the section Homalocarpus. The DNA values obtained appeared to be “quantized”, and data for the majority of species fitted a non-geometrical series with the observed relative terms: 1—8—12—16—20—24—40. The magnitude of these variations in DNA content, the preservation of the karyotype and the tendency towards a simple numerical progression in DNA values, lead us to prefer an interpretation of the evolution of DNA content in terms of differential polynemy to one postulating changes in size of genetic units in an unchanging number of strands per chromosome.

OFF-PEAK ABSORPTION MEASUREMENTS IN FEULGEN CYTOPHOTOMETRY.

The use of off-peak measurements in Feulgen cytophotometry is reported, using intact diploid (x) and tetraploid (y) nuclei of the human anterior pituitary gland as the experimental model studied. Results indicate that the ratio y/x is similar at the 14 wavelengths examined over the range 450 mmicro to 650 mmicro. The value of this ratio was 1.95, falling slightly under the theoretical ratio of 2.0. It was concluded that off-peak absorption measurements are of value in Feulgen cytophotometry. A discussion of the possible justification of off-peak absorption measurements was presented for the case in which the Beer-Lambert relationship was experimentally determined to be followed at a peak wavelength, for all concentrations under discussion, and the curves preceding and/or following the peaks were straight lines coming from a common point. If the curve best fitting the data is a straight line, it follows that the rate of change of absorbance with respect to a linear wavelength scale is constant. This means that for a given increase, or decrease, in wavelength, the same change in absorbance is obtained. From this it follows that absorbance readings at any wavelength in such a region will be equally valid to those taken at the peak. While the finding of such a linear relationship at several concentrations does not guarantee that it will occur at all other concentrations, it is suggestive. The closer the spectra approximate straight lines, the more valid does the use of off-peak measurements become.

The effect of some pituitary hormones on volume and DNA content of cell nuclei of the adrenal cortex in hypophysectomized-castrated rats

1. 1. The volume and the DNA content of the cell nuclei were determined by caryometry and Feulgen cytophotometry in the different layers of the adrenal cortex of the normal rat, the untreated hypophysectomized, castrated rat and the hypophysectomized, castrated rat treated by ACTH, STH and LH. 2. 2. Nuclear DNA content shows a decreasing gradient from the zona fasciculata externa to the zona reticularis. Nuclear volume shows the same pattern except in the zona glomerulosa, where nuclear volume is nearly half the volume of this in the zona fasciculata externa. 3. 3. After hypophysectomy nuclear DNA is decreased in the zona fasciculata externa, but unchanged in the other layers. Nuclear volume is decreased in every layer. 4. 4. Treatment by ACTH normalizes nuclear DNA in the zona fasciculata externa and increases this content in the two inner layers. Nuclear volume shows similar changes. 5. 5. Treatment by STH has no influence on nuclear DNA changes induced by hypophysectomy. Nuclear volume, however, is normalized in the zona glomerulosa and increased in the other layers. 6. 6. ICSH has no influence on the nuclear DNA content, but increases nuclear volume in every layer of the hypophysis-less rat. 7. 7. These results accord with the zonation theory. They support the hypothesis of Deane and Greep, who attribute a glucocorticoid function, depending on ACTH secretion, to the cells of the zona fasciculata externa, while the cell function of the zona glomerulosa is not dependent on ACTH secretion. The cells of the zona fasciculata interna and reticularis seem to be resting cells, which can be activated by high doses of ACTH. 8. 8. The volume of the nucleus is not only dependent on cell function, but also on other factors unrelated to specific cell function such as on the STH secretion of the hypophysis.

The experimental error of Feulgen cytophotometry in the analysis of bull spermatozoa over an extended period of time.

Feulgen cytophotometry has been applied over a period of about two years to the analysis of the deoxyribonucleic acid content of 658 samples of spermatozoa from 275 Santa Gertrudis bulls of the King Ranch, most of which were under two years of age. A system of correction for smear to smear and slide to slide staining variation due to experimental error is presented that restricts the total maximum experimental error of a single determination to 15%, and to 10% in 80% of samples, when 18-25 sperm are measured per smear. Refinements of technique are described by which the maximum error can be reduced to 7% in 95% of samples and to 5% by increase in the number of sperm measured per sample smear.

Differential polyteny and polyploidy in diaspine coccids (Homoptera: Coccoidea).

1. Male germ cells ofPseudaulacaspis pentagona (TARG.), an exceptional species of the coccid subfamilyDiaspinae, contain only approximately half as much DNA (measured by Feulgen cytophotometry) as do those ofChrysomphalus ficusAsh. — a typical representative of the group. 2. Reversing this relationship, the chromosomes ofP. pentagona number 16 in the female, 8 in the male — whileC. ficus has the basic complement of the subfamily, numbering 8 in the female, 4 in the male (Brown andBentsnett 1957). 3. The total mass of the chromosomes in the 2 complements is roughly proportional to the DNA values. It is concluded that 2 downward shifts in the level of chromosomal polyteny have been associated with the doubling of the chromosome number inPseudaulacaspis pentagona. 4. The cytology of the diaspines is discussed in relation to the evolutionary origin of male haploidy in coccids.

Polyteny as a factor in the chromosomal evolution of the Pentatomini (Hemiptera).

1. ThePentatomini, which constitute a single Tribe within one of the Subfamilies of the hemipteran FamilyPentatomidae, include an unusually large number of species. Though these species vary considerably in their size and color, they are very uniform in their essential external and internal morphology. 2. Uniformity also characterizes such features in the Tribe as the chromosome number and meiotic behavior. But in the eleven species here investigated, this uniformity does not extend to the DNA content of the male meiotic cells, as determined by Feulgen cytophotometry. 3. The lowest DNA content is found inThyanta which, in this respect, is well separated from an intermediate group whose DNA values vary around a mean that is roughly double the lowest amount. Some evidence is available that a still higher multiple of DNA may be represented in theAcrosternum-Nezara complex of species. 4. In view of these cytochemical data, the multiplicity of species coupled with a striking uniformity of morphological and cytological features is provisionally explained on the basis of varying polyteny.