Toluidine Blue Test for Sperm DNA Integrity and Elaboration of Image Cytometry Algorithm

Background: Sperm DNA integrity is of paramount importance in the prognosis of fertility. We applied image cytometry to a toluidine blue (TB) test we recently proposed. Methods: Sperm samples from 33 men were assayed for standard sperm parameters and classified as normal or abnormal. Sperm smears were subjected to the TB test, DNA denaturation testing with acridine orange (AO), and terminal deoxyuridine triphosphate biotin nick end labeling (TUNEL). In CCD image analysis, TB-stained sperm cell heads were microscopically assigned to one of four color groups (dark, blue, light violet, and light blue). The optical densities of 6,600 cells in green and red CCD images were used to elaborate an algorithm for discrimination of these groups. Results: The proportions of sperm in TB color groups, as estimated with the developed image cytometry algorithm, correlated with microscopic features. The number of TB dark cells correlated with the number of AO-red and TUNEL+ cells. The proportion of TB dark cells in normal samples did not exceed 35%. Light-blue sperm cell heads prevailed in normal samples, whereas dark and blue sperm cell heads dominated in abnormal samples. Conclusions: The TB test was suitable for the assessment of sperm cell DNA integrity. The elaborated image cytometry algorithm can be used for this purpose and for finer determination of sperm nucleus status.publishersversionPeer reviewe

Consideration on the Metachromatic Spectra of Toluidine Blue Dimers Formed on DNA Oligomers

Metachromatic staining with toluidine blue (TB) has been used as a cytological tool for tissue recognition and cancer diagnosis. Recently, its strong potential for infertility diagnosis was also reported. Metachromatic staining is important in biological applications, but the origin of spectral changes has not been fully understood, although dye aggregation on biological materials is thought to be the cause. In this study, we investigated the dimer structure of TB formed on DNA oligomers by using a computational method, particularly focusing on the spectral changes caused by TB dimer formation. The structure of the TBDNA complexes was constructed on the basis of the calculated molecular structure of TB and crystal data of A-and B-form DNA oligomers, assuming that there was an electrostatic interaction between them. The resulting spectral shift was then evaluated using the extended-dipole model. The examination of B-DNA revealed that possible TB dimers result in a hypsochromic spectral shift of absorption. On the other hand, the dimerization of TB on A-DNA was found to be quite difficult, because the helical geometry of A-DNA restricted the binding sites of TB. These results suggest that the metachromatic color observed during biological staining is significantly affected by the helical geometry of DNA. Toluidine blue (TB) 1 This characteristic makes it possible to stain certain tissue elements in different colors using a single dye. TB, for instance, stains the granules of mast cells and small oral cancers with the metachromatic color purple. 2,3 These color changes have been explained by the dye aggregation formed on biological substrates. The exciton coupling theory 4 states that the quantum mechanical resonance effects in a molecular dimer result in the splitting of the energy level of the monomer excited state into two levels, of which one is more stable and the other is less stable than the monomer. When dye molecules dimerize in a co-parallel arrangement, the electronic transition from the ground state to the less stable upper one is only allowed to give a hypsochromic shift of absorption. TB is known to have three characteristic absorption bands. In a dilute aqueous solution, TB concentration is within 10 ¹5 to 10 ¹6 mol L ¹1 and its absorption maximum appears at around 630 nm. This band, the ¡ band, is believed to originate from the monomer. With an increase in TB concentration the ¡ band shifts to the shorter wavelength region. This band is known as the ¢ band. Further, the absorption peak shifts toward the shorter wavelength regions after adding agar. This is described as the £ band. The ¢ and £ bands have been thought to be caused by the formation of dimers and higher molecular aggregates, respectively.

Differential staining of peripheral nuclear chromatin with Acridine orange implies an A-form epichromatin conformation of the DNA

<p>The chromatin observed by conventional electron microscopy under the nuclear envelope constitutes a single layer of dense 30–35 nm granules, while ∼30 nm fibrils laterally attached to them, form large patches of lamin-associated domains (LADs). This particular surface “epichromatin” can be discerned by specific (H2A+H2B+DNA) conformational antibody at the inner nuclear envelope and around mitotic chromosomes. In order to differentiate the DNA conformation of the peripheral chromatin we applied an Acridine orange (AO) DNA structural test involving RNAse treatment and the addition of AO after acid pre-treatment. MCF-7 cells treated in this way revealed yellow/red patches of LADs attached to a thin green nuclear rim and with mitotic chromosomes outlined in green, topologically corresponding to epichromatin epitope staining by immunofluorescence. Differentially from LADs, the epichromatin was unable to provide metachromatic staining by AO, unless thermally denatured at 94^oC. DNA enrichment in GC stretches has been recently reported for immunoprecipitated ∼ 1Kb epichromatin domains. Together these data suggest that certain epichromatin segments assume the relatively hydrophobic DNA A-conformation at the nuclear envelope and surface of mitotic chromosomes, preventing AO side dimerisation.  We hypothesize that epichromatin domains form nucleosome superbeads. Hydrophobic interactions stack these superbeads and align them at the nuclear envelope, while repulsing the hydrophilic LADs. The hydrophobicity of epichromatin explains its location at the surface of mitotic chromosomes and its function in mediating chromosome attachment to the restituting nuclear envelope during telophase.</p

When three isn't a crowd: a digyny concept for treatment-resistant, near-triploid human cancers

Near-triploid human tumors are frequently resistant to radio/chemotherapy through mechanisms that are unclear. We recently reported a tight association of male tumor triploidy with XXY karyotypes based on a meta-analysis of 15 tumor cohorts extracted from the Mitelman database. Here we provide a conceptual framework of the digyny-like origin of this karyotype based on the germline features of malignant tumors and adaptive capacity of digyny, which supports survival in adverse conditions. Studying how the recombinatorial reproduction via diploidy can be executed in primary cancer samples and HeLa cells after DNA damage, we report the first evidence that diploid and triploid cell sub-populations constitutively coexist and inter-change genomes via endoreduplicated polyploid cells generated through genotoxic challenge. We show that irradiated triploid HeLa cells can enter tripolar mitosis producing three diploid sub-subnuclei by segregation and pairwise fusions of whole genomes. Considering the upregulation of meiotic genes in tumors, we propose that the reconstructed diploid sub-cells can initiate pseudo-meiosis producing two “gametes” (diploid “maternal” and haploid “paternal”) followed by digynic-like reconstitution of a triploid stemline that returns to mitotic cycling. This process ensures tumor survival and growth by (1) DNA repair and genetic variation, (2) protection against recessive lethal mutations using the third genome

Spatial-Temporal Genome Regulation in Stress-Response and Cell-Fate Change

Complex functioning of the genome in the cell nucleus is controlled at different levels: (a) the DNA base sequence containing all relevant inherited information; (b) epigenetic pathways consisting of protein interactions and feedback loops; (c) the genome architecture and organization activating or suppressing genetic interactions between different parts of the genome. Most research so far has shed light on the puzzle pieces at these levels. This article, however, attempts an integrative approach to genome expression regulation incorporating these different layers. Under environmental stress or during cell development, differentiation towards specialized cell types, or to dysfunctional tumor, the cell nucleus seems to react as a whole through coordinated changes at all levels of control. This implies the need for a framework in which biological, chemical, and physical manifestations can serve as a basis for a coherent theory of gene self-organization. An international symposium held at the Biomedical Research and Study Center in Riga, Latvia, on 25 July 2022 addressed novel aspects of the abovementioned topic. The present article reviews the most recent results and conclusions of the state-of-the-art research in this multidisciplinary field of science, which were delivered and discussed by scholars at the Riga symposium