M. G. Manfredi Romanini

The effect of different fixatives on chromatin: Cytochemical and ultrastructural approaches

SummaryThis study explores the effects of two types of fixative on chromatin. The first type (acrolein, glutaraldehyde) engenders a high degree of ultrastructural preservation. The other type are fixatives that are widely used in cytochemistry and cytogenetics (acetic acid, 3∶1 by vol. methanol-acetic acid, methanol alone, formaldehyde).Lymphocytes of adult rats so-fixedin vitro were prepared for electron microscopy or microdensitometric evaluations of smears. Assessments were made of variations in their total protein, nuclear basic protein and DNA contents. DNA was determined both as Feulgen-positive material and by its binding of intercalating dyes (Methyl Green, specific for double-stranded polynucleotides).Our results showed that some fixatives break up the chromatin organization by acting on particular components of chromatin fibres. They can thus be considered to be destructive agentsin situ. In addition, a revaluation of some aldehyde fixatives is proposed for both ultrastructural and cytochemical research.

Expression of cell cycle related proteins—proliferating cell nuclear antigen (PCNA) and statin—during adaptation and de‐adaptation of EUE cells to a hypertonic medium

Abstract. EUE cells adapted to grow for long times in a hypertonic medium have a longer cell cycle than those growing in isotonic medium. To elucidate whether this lengthening involves specific cycle phases to differing extents, the expression of two cycle‐related protein, PCNA and statin, was studied by dual parameter flow cytometry of indirect immunofluorescence protein labelling and DNA content. In isotonic medium, most cells, in all the cycle phases, were PCNA positive; in contrast, PCNA negative cells and statin positive cells were very few in number and only fell in the G0/l range of DNA contents. In hypertonic medium, the frequency of PCNA positive cells was lower, and that of statin positive cells higher, than in isotonic medium, particularly in the Go/1 range of DNA contents: this suggests that a G0 block occurs under long‐term hypertonic stress. Consistently, dual parameter flow cytometric measurement of BrdUrd immunofluorescence labelling and DNA content showed that fewer cells entered S phase in hypertonic medium and their progression through the S phase was slower; evidence was also found for the occurrence of a G2 block. These kinetics changes were fully reversible in isotonic medium, thus indicating the adaptive nature of the EUE response to hypertonicity.

Sperm-chromatin maturation in the mouse

SummaryCytochemical techniques were used to study chromatin during spermiogenesis and sperm maturation in the mouse, starting from the stages at which the substitution of somatic histones by testis-specific proteins occurs. It was possible to distinguish and analyze the different temporal incidence of two processes involved in sperm maturation, i.e. chromatin condensation (a tridimensional highly compacted arrangement) and chromatin stabilization (a tough structure, which protects the genome DNA). The first process, involving a reduction in the nuclear size and a decrease in the amount of sperm DNA accessible to specific cytochemical reactions and stainings, was found to reach its maximum in caput-epididymidis spermatozoa, in which electron microscopy revealed that the sheared chromatin was mainly organized into 120-Å-thick knobby fibers. No further changes were found in sperm up to their appearance in the fallopian tubes. On the contrary, chromatin stabilization, the onset of which occurs in the testis (at the late spermatid stage) via the formation of -S−S- cross-links, is completed in the vas deferens, where chromatin has a superstructure consisting of thicker fibers, with diameters of 210 and 350 Å. The reductive cleavage of disulfides in vas-deferens spermatozoa does not completely destroy the superstructure of sperm chromatin, which could indicate ‘coiling’ of the basic knobby fiber. In fact, when the ion concentration was increased, the chromatin of vas-deferens spermatozoa appeared to be organized into fibers with diameters similar to those of the caput epididymidis. This unique organization of mature sperm chromatin should have an essential role in the fast swelling of spermatozoa during fertilization.

Effect of hypertonic medium on human cell growth: III. Changes in cell kinetics of EUE cells

The effects of hypertonicity on cell kinetics of EUE cells in culture have been investigated. After 4 days of growth in a hypertonic medium, the plating efficiency of EUE cells was reduced and cell growth was significantly slowed. Flow cytometric measurements of DNA content in synchronized cells, as well as flow cytometric determinations of DNA content and bromodeoxyuridine incorporation in asynchronous cells, also showed that the cell cycle is slowed in a hypertonic medium. In addition, the fraction of cycling cells is smaller and their progression through the S phase slower than in an isotonic medium.

Feulgen-DNA Content and C-Banding of Robertsonian Transformed Karyotypes in Dugesia Lugubris

SUMMARYIn the planarian species, Dugesia lugubris, two biotypes are found: E (2n = 8, n = 4) and F (2n = 6, n = 3); on the basis of karyometric studies it has been hypothesized that the second was derived from the first through a Robert-sonian mechanism of centric fusion. The quantitative cytochemical data reported here confirm the hypothesis of karyotype evolution, since there are no significant differences between the DNA content of the nucleus in the two biotypes. The regenerative blastemas of both biotypes contain a number of cellular populations with a variable Feulgen-DNA content; these correspond to successive doublings of the 2C diploid content. In addition, metaphase plates with multistranded chromosomes have been found. A difference between the chromosome C-banding in the two biotypes has also been observed.

Feulgen-DNA amounts and karyotype lengths of three planarian species of the genusDugesia

Genome sizes of the planariansD. lugubris (2n=8),D. polychroa (2n=8) andD. benazzii (2n=16) were evaluated on metaphase plates by measuring both the Feulgen-DNA contents and the karyotype lengths. In the three species, genome sizes are significantly different; this finding rules out the possibility of a karyotype evolution through simple chromosome rearrangements betweenD. lugubris andD. polychroa. A different Feulgen-DNA content per unit length of karyotype in the three species studied was also found, which suggests that DNA could be differently packed along metaphase chromosomes.

Evolutionary implications of the analysis of feulgen hydrolysis curves in mammalian species with a different nuclear DNA content

The nuclear DNA or DNP differences in hydrolysis resistance are utilized here as an indication of the greater or lesser complexity of DNA-protein bindings, possibly related to the degree of heterochromatinization. They have been studied with the Feulgen reaction in lymphocytes of peripheral blood smears of Cercopithecus aethiops aethiops (Primates) (a species in which adaptive evolution still appears to be under way) and of Rhinolophus ferrumequinum (Microchiroptera), a representative of an older group of Mammals, Chiroptera, which is considered as evolutionarily established at least since the Eocene period (Simpson, 1945). These species have a very different “genome size” (recorded as Feulgen-DNA nuclear content): the percent ratio is at least 130:100 (Capanna & Manfredi Romanini, 1971) . From the comparison between the two hydrolysis curves it appears that their general shape is similar and that the height differences are constant for each hydrolysis time, with the exception of the first peak which is operationally defined as the acid labile fraction of DNA. This peak is higher in C. aethiops aethiops than in R. ferrumequinum. Both curves fall short of characteristic platform (120 min — 360 min) of “acid resistant” DNA, which is constantly recorded in Homo and tentatively attributed to some particular DNA-protein link (depolymerization peak of Anderson et al., 1971, 1975). No qualitative difference is remarkable in the DNA protein ratio, which could be relied to heterochromatinization materials, or to a selective loss of heterochromatic fractions of chromatin. We are therefore lead to interpret the present data as a verisimilar indication that Chiroptera ancient specialization is ascribable to a mechanism of evolutionary loss which, unlike the actual microevolution of some species of Mammals, could not be preceded by a selective heterochromatinization.

Genome size and «C-heterochromatic-DNA» in man and the african apes

The genome sizes and the amounts of DNA after C-banding pretreatments (C-heterochromatic DNA) were measured by quantitative cytochemical methods in man and the African apes,Gorilla gorilla andPan troglodytes. As evaluated by flow cytometry on propidium-iodide-stained lymphocytes, gorilla and chimpanzee have genome sizes larger than man. On the basis of the different resistance of metaphase chromosome DNA to the C-banding procedure, two genome compartments were defined, i.e.,C-heterochromatic-DNA andeuchromatic-DNA. The latter proved to be fairly constant in man and the African apes (as well as in two hylobatid species), whereas the variable amounts ofC-heterochromatic-DNA account well for the interspecific differences of genome size among the hominoid species studied so far. During karyotype diversification, quantitative changes (with either gains or losses) ofC-heterochromatic-DNA seem to have taken place independently in the hylobatid and the man/African ape lineages.

Osservazioni sul contenuto in ADN dei nuclei degli eritrociti dei ciprinidi

Summary The results obtained histophotometrically on the nuclear content of DNA of erythrocytes in 20 different 6pecies of. Cypriuidae are presented and discussed as to the possible existence of cases of polyploidism in the famili of Cypriuidae.

Histochemistry: work in progress

As our subscribers have been notified in a recent letter, this issue of the European Journal of Histochemistry is the last one to be edited and printed by “Luigi Ponzio e figlio editori”: They have generously collaborated with us with the greatest care and professionality for the past twenty years and for that we wish to thank Mr. Luigi Ponzio and his entire staff (in particular, Mr. Luigi Guardamagna) most hearthly. The new publisher Tipografia PIME Editrice has assured us of a strong support in the promotion and the development of the image of the European Journal of Histochemistry in its relations with the media at all levels. While a change in the Publisher may be seen as a merely technical one, it is also the occasion to reflect upon how a Journal such as ours can be enriched. The Journal’s topics touch upon many of the basic issues in biology and, through these, in pathology. Our subjects are humans, as well as all animals and plants, not only looked upon as objects for comparison and taxonomic analysis, but also as models for detailed study