Cytogenetic analysis of day-7 bovine embryos by cytophotometric DNA measurements.

A Feulgen staining procedure that stains the DNA of individual fixed nuclei stoichiometrically was used to analyse cytophotometrically the incidence of total ploidy and mixoploidy in 28 day-7 bovine embryos that had been fixed after collection ('non-cultured' embryos). The influence of culture on the incidence of chromosome abnormalities was further studied in another group of 24 embryos ('cultured' embryos) by culturing them for 24 h in Whittingham's medium. Of the total 52 embryos studied, two appeared to be entirely abnormal: one embryo was completely haploid, whereas the other embryo was completely triploid. Individual hyperdiploid nuclei and hypodiploid nuclei were frequently observed in the otherwise diploid embryos. As haploid polar bodies can still be present in morulae and blastocysts (to a maximum of three), only embryos with more than three hypodiploid nuclei were considered as abnormal. Of the 'non-cultured' embryos, 33.3% had one or more hyperdiploid nuclei, whereas 51.9% had more than three hypodiploid nuclei. In this latter group, 35.7% of the embryos also had hyperdiploid nuclei. The results also showed that day-7 bovine embryos that are completely haploid, completely triploid or mixoploid cannot be detected only by examining their morphology. It is concluded that the incidence of, especially, mixoploidy in embryos can be better studied by measuring the DNA content of the individual nuclei of an embryo rather than by analysing chromosomes, as in the latter method only dividing cells can be analysed. The presence of hyperdiploid and hypodiploid nuclei may indicate the frequent occurrence of mitotic segregation failures during mitosis in bovine embryos.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for diploidy in metacyclic forms of African trypanosomes.

The DNA contents of bloodstream form trypanosomes (life cycle stages circulating in the blood of the vertebrate host) of four African Trypanosoma species and of metacyclic forms (the life cycle stage that is injected into the vertebrate by the tsetse fly during its bite) of the same four species were measured by cytofluorometry of individual cells or nuclei. The results showed unambiguously that the metacyclic forms cannot be considered to be products of meiosis containing only half of the DNA of bloodstream forms, in contrast to what was previously reported for Trypanosoma brucei [Zampetti-Bosseler, F., Schweizer, J., Pays, E., Jenni, L. & Steinert, M. (1986) Proc. Natl. Acad. Sci. USA 83, 6063-6064] during an attempt to localize the gametes in the life cycle after experimental evidence of sexual gene exchange in this parasite was reported.

DNA content and structure of (double) minutes of a methotrexate-resistant cell line.

We have determined the DNA content of intact double minutes (DMs) and of single minutes (SMs) by fluorometry of the individual chromatin bodies in metaphase spreads after staining with Feulgen-Schiff pararosaniline. We find that the intact DMs and SMs of the methotrexate-resistant mouse cell line 3T6R50 contain 4.4 megabase pairs (Mb) and 2.6 Mb DNA respectively, using the DNA content of E. coli (4.7 Mb) as a reference. As the pulsed field gradient gel electrophoresis experiments by van der Bliek et al. (1988) have indicated that the minutes of 3T6R50 cells contain a homogeneous population of 2.5 Mb DNA circles, we conclude that a SM contains one circular double strand DNA molecule of approximately 2.5 Mb, whereas DMs contain two.

Flow cytometric determination of carbohydrates in human erythrocytes.

Carbohydrate components known from biochemical analysis to be present in peripheral normal human erythrocytes so far could not be detected cytochemically. By periodic acid oxidation followed by Schiff pararosaniline (SO2) staining, however, a specific fluorescent signal can be obtained, strong enough to allow measurement by flow cytometry. Dimethylsuberimidate fixation results in low autofluorescence and low staining of unoxidized cells. By treating erythrocyte ghosts similarly, it is found that about 20% of the signal is present in the membrane, most probably due to glycophorins. The main signal resides in the matrix of the fixed erythrocyte and may be due to traces of glycogen and to the glycosylation of proteins, especially hemoglobin.

Matrix models. Essential tools for microscopic cytochemical research.

An overview is given of the preparation and use of model systems for cytochemistry, dealing with quantitative as well as qualitative aspects. Descriptions are given of the various possibilities to prepare cytochemical matrix models, ranging from macroscopic and microscopic films, to models with more cell-like dimensions as agarose beads, artificial cells and erythrocyte ghosts. Such models allow the study of a large variety of cytochemical processes. Their potentialities are demonstrated in a number of specific applications, comprising: the study of the influence of fixation on cellular processes, reaction specificity and reaction kinetics, quality of reagents and biochemical calibration in cytochemical staining; factors influencing localization of the specific endproduct in enzyme cytochemistry; immunocytochemistry and hybridocytochemistry.

Optical errors in scanning stage absorbance cytophotometry. II. Application of correction factors for residual distributional error, glare and diffraction error in practical cytophotometry.

In a preceding article theoretical methods were derived for correcting the integrated absorbance values of microscopic objects determined with a scanning stage cytophotometer for the systematical erros due to residual distributional error, diffraction error, and glare error. For an experimental investigation of these results, the local apparant transmission at the center of an opaque particle was determined and this value used as a measure for the substage glare. By sufficient reduction of the size of the illuminated field, this substage glare could be kept below 1% in our scanning stage cytophotometer. The magnitude of the diffraction error was experimentally approached by comparing the values found for the integrated absorbance of the same amount of chromophore, dispersed over two different areas by crushing or centrifugation. After appropriate correction for the residual distributional error, the remaining difference was ascribed to the diffraction error, caused by diffraction at the edges of the object, and to the glare error, present all over the measured area. The local borderline corrections found necessary to obtain the best matching corrected integrated absorbance values were between 3 and 5%, in good agreement with the value derived theoretically. The importance of these corrections for the determination of the integrated absorbance of common biological objects is discussed.

A theoretical study of concentration of profiles of primary cytochemical-enzyme reaction products in membrane-bound cell organelles and its application to lysosomal acid phosphatase.

A numerical method was developed for computing the steady-state concentration gradient of a diffusible enzyme reaction product in a membrane-limited compartment of a simplified theoretical cell model. In cytochemical enzyme reactions proceeding according to the metal-capture principle, the local concentration of the primary reaction product is an important factor in the onset of the precipitation process and in the distribution of the final reaction product. The following variables were incorporated into the model: enzyme activity, substrate concentration, Km, diffusion coefficient of substrate and product, particle radius and cell radius. The method was applied to lysosomal acid phosphatase. Numerical values for the variables were estimated from experimental data in the literature. The results show that the calculated phosphate concentrations inside lysosomes are several orders of magnitude lower than the critical concentrations for efficient phosphate capture found in a previous experimental model study. Reasons for this apparent discrepancy are discussed.

The influence of chromatin compactness on the stoichiometry of the Feulgen-Schiff procedure studied in model films. I. Theoretical kinetics and experiments with films containing isolated deoxyribonucleic acid.

Theoretical considerations on the expected kinetics of the course of the Feulgen-Schiff reaction show that the leveling off of the first part of the Feulgen hydrolysis curve can be explained by the gradual conversion of deoxyribonucleic acid (DNA) to apurinic acid (APA). In addition, depolymerization of DNA caused by the acid used for hydrolysis can account for the decline after a maximum is reached in this curve. With the aid of polyacrylamide model films containing DNA, a detailed study was made both of the process of purine liberation which results in the formation of APA and of the depolymerization processes which cause losses of stainable material. The liberation of purine bases was analyzed by ultraviolet absorbance measurements and by gel chromatography of the neutralized hydrolysing acid. APA concentration was monitored by following the loss of ultraviolet absorbance associated with the purine losses. The depolymerization process was followed by phosphorus determinations. The experimental results were found to be in accordance with the kinetics expected from the theoretical model.

The influence of chromatin compactness on the stoichiometry of the Feulgen-Schiff procedure studied in model films. II. Investigations on films containing condensed or swollen chicken erythrocyte nuclei.

As models for different states of chromatin compactness, nuclei from chicken erythrocytes were isolated and either osmotically swollen or kept as condensed as possible. Both types of nuclei were then fixed and incorporated into polyacrylamide films. Hydrolysis with 5 N HCl and staining with Schiff's reagent of these model films were studied using several parameters. The phosphate content of the films was analyzed as a parameter for the depolymerization losses and the staining with Schiff's reagent as a parameter for the apurinic acid (APA) content. The loss of ultraviolet absorbance from the films and the accumulation of ultraviolet absorbing substances in the hydrolyzing acid were monitored as parameters for the progress of hydrolysis. Conversion of the generated aldehyde groups to APA-Schiff chromophore is shown to take place with the same stoichiometry for both types of nuclei as well as for DNA in model films. It is further shown that the nuclei- and DNA-films are suitable models for investigating the influence of chromatin compactness on the course of the Feulgen-Schiff reaction. For the most compact form of chromatin studied, a very high reduction in staining intensity of up to 40% could be demonstrated after certain normally applied hydrolysis times. This is due primarily to a decrease with a factor of 2.3 of the depurination rate constants of these models (from 0.030/min to 0.013/min). Therefore prolonged hydrolysis periods are required to obtain the same APA concentrations, but then depolymerization processes cause losses of nuclear material. The differences in depurination rates could be explained by a decrease in [H3O]+ in the neighborhood of the purine-sugar linkages, caused by the presence of fixed positive charges form the protein components of the chromatin. These findings may explain the cytophotometrically determined differences in chromophore yield of 10-20% found in the nuclei of cells with different states of compactness of their chromatin. The descending part of the Feulgen hydrolysis curve represents the depolymerization of APA and loss by diffusion of the reaction products. In the Appendix, cytophotometric data of cells have been analyzed to show that this part of the hydrolysis curve may be used to estimate the acid stability of chromatin complexes. The depurination and depolymerization rates found closely correspond with the data obtained from the model films.

The interaction of apurinic acid aldehyde groups with pararosaniline in the Feulgen-Schiff and related staining procedures.

The equilibrium reactions involved in the formation of the apurinic acid (APA)-Schiff chromophores in the staining phase of the Feulgen-Schiff reaction do not allow a quantitative conversion of APA to these chromophores. By modification of the sulfite and dye concentrations and the pH of the staining reagents, or by using better solvents for pararosaniline like acetic acid or dimethylsulfoxide (DMSO) a shift of these equilibria was attempted in order to obtain a higher amount of APA-bound dye. A 40% higher absorbance, when compared with the normal Schiff-staining, was obtained in model films by staining with a saturated solution of pararosaniline in a 1:1 v/v mixture of DMSO and SO2-water, followed by rinsing in SO2-water. A doubling of the absorbance resulted in the same objects when a saturated solution of pararosaniline in a 2 M acetic acid/acetate buffer of pH 4.45 was used for staining, followed by a short rinse in SO2-water. Amino groups (as found in histones) are shown to compete with the amino groups of pararosaniline for the APA aldehydes. This effect, although causing lower staining intensities, is shown not to be the explanation for the differences in stain content found between more and less compact forms of chromatin. Depending on the pH, and dye and sulfite concentrations of the staining reagents, the following components are considered as possible contributors to the mixture of chromophores (Duijndam et al., 1973 b) formed between APA and Schiff's reagent or its modifications: 1. An acid labile component with a wavelength of maximal absorbance (lambda max) near 510 nm; its structure is probably the azomethine--CH=N--; 2. A relatively acid stable component with a high value of molecular absorbance (epsilon), an lambda max near 570 nm and possibly having an enamine structure--CH=CH--NH--; 3. A component with intermediate acid stability, low epsilon, and lambda max near 540 nm, and which is probably an alkylsulfonic acid --CH(SO3H)--NH--compound. Small differences in the staining conditions in the histochemical application of the Feulgen-Schiff reaction may cause a shift in the ratio between especially components 2 and 3, resulting in variations in stain content and in lambda max.