Introduction. In the last decade, an understanding of the two-fold nature of genetic apparatus damage in spermatozoa has emerged: abnormal chromatin condensation (“immature” chromatin, ICH) related to defective protaminization and leading to altered epigenetic regulation of the early embryogenesis, and disruption of DNA integrity, i. e. DNA fragmentation (DFS).Objective. Study of the correlation between abnormal chromatin condensation in spermatozoa and DFS.Materials and methods. The study included spermatozoa of 54 fertile males (1st group, control), 46 patients with primary infertility (2nd group), and 111 patients whose wives had a history of pregnancy abnormalities or failures of the assisted reproductive technology (ART), i. e. arrested embryonic development (3rd group). Presence of ICH was identified by quantitative electron microscopy, presence of DFS by TUNEL. Study of ICH and DFS in the same spermatozoon was conducted using correlation microscopy (TUNEL with subsequent ultrastructural analysis of the labeled cells).Results. The number of ICH spermatozoa significantly differed in the 3rd group from the control group (29.26 ± 13.49 vs. 22.43 ± 9.54; p = 0.006). The number of ICH spermatozoa in the 2nd group was higher than in the control group, but the difference wasn’t statistically significant (p = 0.061). A significant difference in the number of spermatozoa with residual cytoplasm on the head was observed between the fertile group and 2nd and 3rd groups (p = 0.0001 and p = 0.0006, respectively) and on the neck (p = 0.0002 and p = 0.0003, respectively). Number of DFS spermatozoa in the second group significantly differed from the control (21.40 ± 11.88 vs. 13.70 ± 7.00, p = 0.03), but this difference wasn’t observed for the 3rd group. A very weak correlation between the number of DFS and ICH spermatozoa was observed in all three groups (r = 0.18, r = 0.33, and r = 0.01, respectively). Forty-six (46) spermatozoa were studied using correlation microscopy, among them 23 were DFS and 23 were ICH. In 11 of 23 HDS spermatozoa DFS was observed. In spermatozoa with condensed chromatin, DFS was identified in 6 spermatozoa.Conclusion. DFS and abnormal chromatin condensation are highly independent parameters, which should be considered during identification of the cause of infertility. ICH is more frequent in spermatozoa of patients whose wives have a history of embryogenesis abnormalities. DNS is more frequent in primary infertility.

Using the methods of light microscopy and DNA cytophotometry, structural organization of the calli of the barley hybrid line Duet x Bios grown in the absence and in the presence of aluminum ions (20 and 40 mg/L) was studied. In the morphogenic calli grown in the standard conditions without aluminum, three main cell types were identified: meristematic, differentiated, and dying. Meristematic cells form a morphogenic zone. A characteristic feature of differentiated cells is a progressive accumulation of vacuoles with storage substances. Structurally separated zones of meristematic cells were not found the calli surviving in the presence of aluminum. Besides, the number of differentiated cells with storage substances is dramatically reduced and the number of dying cells is increased in such calli. According to the cytophotometry data, morphological changes in the aluminum-tolerant calli are accompanied by partial suppression of proliferation, accumulation of cells in postsynthetic phase of the cell cycle, and polyploidization, presumably caused by the DNA endoreduplication. It is noteworthy that the aluminum-tolerant calli exhibit a high regenerative capacity: the proportion of morphogenic calli surviving in the presence of 40 mg/L aluminum is about 5 times greater than that in the calli grown in the control medium. To identify cellular targets of the aluminum effect in differentiated tissues, the roots of rhizogenic calli were studied. For this purpose, the calli derived from aluminum-tolerant plants (cultivar Kupetz), from aluminum-sensitive line 999-93, and from aluminum-tolerant line 917-1 obtained by the method of cell selection were used. It was found that aluminum induces the death of differentiated cells of epidermis and vascular system ingrowing roots of calli derived from non-tolerant lines, whereas undifferentiated cells of meristem remain undamaged. In contrast, rhizogenic calli derived from tolerant forms produce normal roots. This observation has an important prognostic value because it makes it possible to select aluminum-tolerant regenerants at the early stages of cell selection. The data suggest that most likely cause of the emergence of resistant lines is somaclonal variation induced by high concentrations of the selecting agent. Under stressful conditions such variability may result in the in vitro system from the activation of epigenetic factors, such as, for example, methylation of DNA or modification of the chromatin proteins.

In determination of salt resistance of wild plants, cytophotometry has been used for evaluation of distribution of nuclei of the root meristem cells. Salt resistant and salt sensitive plants of two Thellungiella species whose seeds were collected at the places of their vegetation, as well as of two Aegilops L. species from the collection of the All-Russia Research Institute of Plant Industry (VIR), have been analyzed in the experiments. Salt resistant cells of Thel. botschantzevii and Ae. taushii (k-677) were accumulated in the G1 period of the interphase, which indicates high adaptation of these plants to high concentrations of salts. It has been demonstrated that cytophotometry can be used for determination of salt resistance of wild plants growing in the climatic zones appropriate for their natural propagation.

In this study, the morphological and cytoembryological analyses of the tomato plants transformed with the genes encoding chitin-binding proteins (ac and RS-intron-Shir) from Amaranthus caudatus L. and A. retroflexus L., respectively, as well as the gene amp2 encoding hevein-like antimicrobial peptides from Stellaria media L., have been performed. The transgenic lines were adapted to soil and grown in the greenhouse. The analysis of putative transgenic tomato plants revealed several lines that did not differ phenotypically from the wild type plants and three lines with disruption in differentiation of the inflorescence shoot and the flower, as well as the fruit formation (modified plants of each line were transformed with a single gene as noted before). Abnormalities in the development of the generative organs were maintained for at least six vegetative generations. These transgenic plants were shown to be defective in the mail gametophyte formation, fertilization, and, consequently, led to parthenocarpic fruits. The detailed analysis of growing ovules in the abnormal transgenic plants showed that the replacement tissue was formed and proliferated instead of unfertilized embryo sac. The structure of the replacement tissue differed from both embryonic and endosperm tissue of the normal ovule. The formation of the replacement tissue occurred due to continuing proliferation of the endothelial cells that lost their ability for differentiation. The final step in the development of the replacement tissue was its death, which resulted in the cell lysis. The expression of the genes used was confirmed by RTPCR in all three lines with abnormal phenotype, as well as in several lines that did not phenotypically differ from the untransformed control. This suggests that abnormalities in the organs of the generative sphere in the transgenic plants do not depend on the expression of the foreign genes that were introduced in the tomato genome. Here, we argue that agrobacterial transformation affects, directly or indirectly, expression of genes encoding for transcription factors that can activate a gene cascade responsible for the normal plant development.

Interphase prenucleolar bodies are globular bodies which accumulate in large numbers in the nucleoplasm of cultivated cells after hypotonic treatment and subsequent return to isotonic conditions; detailed studies of the role of these structures in the recovery of the nucleolus have not yet been performed. The limited mobility of interphase pronucleoli within the nucleus has been demonstrated. Exchange of the major nucleolar protein B23 between prenucleolar bodies and the surrounding nucleoplasm, rather than stable binding of this protein to the prenucleolar bodies, has been demonstrated using fluorescence recovery after photobleaching method. Gradual accumulation of B23 in the recovering nucleolus with concomitant disappearance of prenucleolar bodies has been demonstrated.

Changes in chromatin structure at different stages of differentiation of human spermatids were studied. It was shown that, in nuclei of early spermatids, chromatin is loosely packed and its structural element is an 8-nm fiber. This “elementary” fiber is predominant at the initial stages of differentiation; in the course of maturation, it is replaced by globular elements approximately 60 nm in diameter. In intermediate spermatids, these globules start to condense into fibrillar aggregates and reduce their diameter to 30–40 nm. At all stages of spermatid maturation, except the final stages, these globules are convergence centers for elementary fibers. This remodelling process is vectored and directed from the apical (acrosomal) to the basal pole of the nucleus. In mature spermatids, the elementary 8-nm fibers are almost absent and the major components are 40-nm fibrillar aggregates. The nuclei of mature spermatids are structurally identical with the nuclei of spermatozoa with the so-called “immature chromatin,” which are commonly found in a low proportion in sperm samples from healthy donors and may prevail over the normal cells in spermiogenetic disorders. The cause of this differentiation blockade remains unknown. Possibly, the formation of intermolecular bonds between protamines, which are required for the final stages of chromatin condensation, is blocked in a part of spermatids. The results of this study are discussed in comparison with the known models of nucleoprotamine chromatin organization in human spermatozoa.

Dynamics of an antipodal complex formation in wheat ( Tritiñum aestivum L.) has been observed in detail using a reconstruction of serial semifine sections. Three consecutive crucial stages have been identified in the development of the antipodal complex: (1) proliferation of initial cells, (2) growth and functional differentiation of antipodal cells, and (3) cell apoptosis. Specific features of the mitotic division of antipodal cells have been characterized. It has been shown that the structure of interphase nuclei and mitotic chromosomes of proliferating antipodal cells is similar to that of nucellar cells surrounding the embryo sac. According to the reconstruction of appropriately oriented serial sections, the division of antipodal cells is asynchronous. DNA content in differentiated antipodal cells has been determined by a cytophotometric analysis; in the case of a mature embryo sac, the ploidy of antipodal cells varied from 8 to 32C. Proliferation and DNA endoreduplication processes in the antipodal complex proceed at different time; the second process starts only after the termination of the first one. DNA endoreduplication is accompanied by total chromatin remodeling; as a result, giant chromosomes are formed in the nuclei of antipodal cells. The final stage of the antipodal complex development is programmed cell death or apoptosis. A model for the structural organization of an antipodal complex has been proposed based on the layer arrangement of cells. The secretory activity of antipodal cells directed towards the endosperm syncytium has been detected for the first time. The analysis of “truncated” ovules with an undeveloped endosperm has shown that developing endosperm can be a possible inductor, which stimulates the functional activity of antipodal cells and triggers their terminal differentiation. The obtained results evidence the functional role of antipodal cells in the development of the endosperm and embryo.

The structural organization of roots, induced in the callus cultures of barley genotypes differing in aluminum tolerance: cv Kupetz, cv 999-93 and its somaclonal form R 999-93 was studied. The goal of this research was to determine how the toxic effects of aluminum at low pH affects the structure of the root and any cells damage of different tissues meristematic zone. At pH 5.8–6.0 root callus from different genotypes had specific features, manifested in the structure root cap, the epidermis, the form of roots. In the presence of 40 mg/1 Al3+ ions and pH 3.7–3.8 in the roots, callus was obtained from the cv Kupetz, there is strong damage to the epidermis, root cap and some other tissues, which, although and resulted in the death of cells, however, were not lethal character. In roots, callus derived from varieties 999-93, under the influence Al3+ ions was observed irreversible changes that lead either to compaction cytoplasm and nuclei, especially in the central part of the root or to the plasmolysis and the destruction of cells, especially in the meristematic zone. On transverse sections was found damaged cells of the central part of the root, formation of the densified outer cell wall of epidermal cells. At the investigation of R 999-93 callus roots it was be shown that most of the cells of meristematic zone of root retained their viability at influence of Al3+ ions. So, the somaclon from cv 999-93, obtained earlier in callus culture by selection on Al-tolerance, differs from the original form at the level of mesostructure.

In this work we analyzed the results of the transformation of tomato plants Solanum lycopersicum by gene FeSOD1 from Arabidopsis thaliana, equipped with the signal sequence for targeting into chloroplasts. PCR analysis showed that the gene was integrated into the genome of several tomato plants that underwent transformation followed by selection in the kanamycin-containing medium. Two lines, provisionally denoted as nos. 6 and 8, were selected from the independent transformants. Line 6 was characterized by a reduced growth rate and altered leaves and line 8, by normal growth and leaves typical for control plants. Both lines showed a significant increase in SOD activity. In line 8 the increase in SOD activity was accompanied by an increase of ascorbate peroxidase activity, and in line 6 this effect was not present. Electron microscopic analysis of parenchymal and guard cells of both lines was performed, with an emphasis on the ultrastructural organization of chloroplasts. It is shown that the chloroplasts of the two transgenic lines differ in the number and size of starch grains and deposited plastoglobules as well as in the organization of lamellae and grana. Taken together, the results indicate that the expression of the introduced gene FeSOD1 has a significant effect on metabolic processes in the plastids. The findings are discussed in relation to the hypothesis about the importance of low concentrations of ROS for the integration of structure and function of chloroplasts.

Nonalcoholic steatohepatitis in rats induced by the high-fat diet was used as an experimental model for testing hepatoprotective properties of drug based on water-ethanol extract from oyster mushroom mycelium. Progression of pathology development was monitored histologically and by biochemical assay of blood samples. Subcellular response was analyzed using electron microscopy. It was shown that administration of the high-fat diet for 14 days leads to a development of adipose degeneration of liver (steatosis). Histological evidences were confirmed biochemically. Alkaline phosphatase, bilirubin, free cholesterol and lowdensity lipoprotein cholesterol levels were increased in blood of experimental animals in comparison to control. Administration of the drug was performed in parallel with high-fat food. It was shown that pathological alterations of liver in this case were reduced at both organ and cellular levels. Cholesterol and triglyceride levels were close to those in control animals. The data obtained confirm that the drug assayed can be used in clinical practice for prevention and treatment of nonalcoholic steatohepatitis.

The structural organization of the nuclear matrix of pericentromeric heterochromatin blocks (chromocenters) was examined in cultured murine fibroblasts. After 2 M NaCl extraction without DNase I treatment, chromocenters became extremely swollen and could not be recognized with conventional electron microscopy. Using immunogolding with anti-topoisomerase IIα antibodies, we demonstrated that residual chromocenters were divided into numerous discrete aggregates. After 2 M NaCl extraction with DNase I treatment, the residual chromocenters looked as the dense meshwork of thin fibers and, therefore, were easily distinguished from the rest of nuclear matrix. Extraction with dextran sulfate and heparin resulted in chromocenter decondensation. Chromatin complexes with rosette organization (central core from which numerous DNA fibers radiated) were seen. Most likely, the appearance of these rosettes was a consequence of incomplete chromatin extraction. Thus, the nuclear matrix of pericentromeric chromosome regions in cultured murine fibroblasts is morphologically distinguished from the rest of the nuclear matrix.

Substantial differences are revealed by the transmission electron microscopy method in the ultrastructure of cell compartments containing storage reserves in plants obtained by cell selection and control plants germinated against the background of the action of NaCl and an osmotic.

The nuclear pore complexes are complex protein structures located in the nuclear envelope, where they control the nuclear-cytoplasmic transport, and inside the stacks of endoplasmic reticulum cisternae, annulate lamellae. After overexpression of some nucleoporins, numerous granules are visible in the cytoplasm. According to the published data, these granules are the annulate lamellae. In the current paper, the structural organization of POM121-containing granules was analyzed using correlative light and electron microscopy. The ultrastructural study demonstrates that POM121-containing granules are not annulate lamellae but aggregates of endoplasmic reticulum membranes. Thus, overexpressed POM121 is not able to induce the annulate lamella formation. The mechanisms of self-organization of non-functional structures (such as the aggregates of endoplasmic reticulum membranes described here) and possible involvement of these mechanisms in the formation of cellular structures are discussed.

According to the radial loop model of chromosome organization, a major role in the formation and maintenance of chromosomes is played by the residual structures (the nuclear matrix in interphase nuclei and the chromosome scaffold in metaphase chromosomes). However, in vivo microscopy has recently revealed that the components of these "static" structures are highly mobile and continuously exchanged between specific target sites and the nucleoplasm or cytoplasm. This contradiction between predicted stability and observed dynamics led us to reexamine the principles underlying the association of proteins with residual structures. In the present paper, we have analyzed the association of two perichromosomal layer proteins, pKi-67 and B23, with the residual structures. The results show that these two proteins are associated with residual structures throughout the cell cycle; only those structures change that contain proteins precipitated by 2 M NaCl (nucleoli, perichromosomal layer, prenucleolar bodies, cytoplasm of mitotic cells). Both pKi-67 and B23 remain associated with the nuclear matrix even when they are translocated to nucleoplasmic foci due to inhibitor action or hypotonic treatment. However, in most cases it remains possible to extract a structurally visible protein fraction with 2 M NaCl (protein distributed in nucleoplasm). One may suppose that the protein fraction associated with residual structures includes molecules interacting with their binding sites at the moment of permeabilization, while the free proteins are extracted (i.e., during the interaction with binding sites, these proteins form salt-resistant complexes; however, on diffusion the same proteins are extractable by the high-salt solution). The residual structures may be considered as a "snapshot" of all proteins transiently (or statically) bound to their target sites at the moment of permeabilization.

Influenza A virus matrix M1 protein is membrane associated and plays a crucial role in virus assembly and budding. The N-terminal two thirds of M1 protein was resolved by X-ray crystallography. The overall 3D structure as well as arrangement of the molecule in relation to the viral membrane remains obscure. Now a proteolytic digestion of virions with bromelain was used as an instrument for the in situ assessment of the M1 protein structure. The lipid bilayer around the subviral particles lacking glycoprotein spikes was partially disrupted as was shown by transmission electron microscopy. A phenomenon of M1 protein fragmentation inside the subviral particles was revealed by SDS-PAGE analysis followed by in-gel trypsin hydrolysis and MALDI-TOF mass spectrometry analysis of the additional bands. Putative bromelain-digestion sites appeared to be located at the surface of the M1 protein globule and could be used as landmarks for 3D molecular modeling.

The extensive use of herbicides in agriculture becomes an important factor in environmental pollution, especially in case of slowly degradable compounds. Some agents act on plants during a long period of time, even if a very low concentration of the herbicide remains in the soil. Here, we investigated the tox-icological effect of a low concentration of dinitroaniline herbicide, trifluralin, on growing seedlings of Hordeum vulgare L. Trifluralin in concentration of 1 μg/ml inhibited root growth. The mitotic activity of meristematic cells was suppressed due to the retardation of metaphase progression — alteration that can be caused by cytoskeleton disorder. Using antibodies to a-tubulin, we investigated the distribution of microtubules in root meristem cells. During all stages of mitosis, the highly regular system of micro-tubular cytoskeleton observed in control cells was slightly disorganized. An examination of root structure using light and electron microscopy demonstrated that the cell walls did not form normally during cell division that led to the appearance of large multinucleated cells. Also, the premature (pathological) cell differentiation was induced by trifluralin. A part of differentiating cells showed intracellular structural changes that are consistent with programmed cell death. It seems that the development of alterations in trifluralin-treated roots was due to the microtubular cytoskeleton disorganization.

The goals of the study were: (1) to explore the communication between human mesenchymal stem cells (MSC) and rat cardiac myocytes resulting in differentiation of the stem cells and, (2) to evaluate the role of mitochondria in it. Light and fluorescence microscopy as well as scanning electron microscopy revealed that after co-cultivation, cells formed intercellular contacts and transient exchange with cytosolic elements could be observed. The transport of cytosolic entity had no specific direction. Noticeably, mitochondria also could be transferred to the recipient cells in a unidirectional fashion (towards cardiomyocytes only). Transmission electron microscopy revealed significant variability in both the diameter of intercellular contacting tubes and their shape. Inside of these nanotubes mitochondria-resembling structures were identified. Moreover, after co-cultivation with cardiomyocytes, expression of human-specific myosin was revealed in MSC. Thus, we speculate that: (1) transport of intracellular elements to MSC possibly can determine the direction of their differentiation and, (2) mitochondria may be involved in the mechanism of the stem cell differentiation. It looks plausible that mitochondrial transfer to recipient cardiomyocytes may be involved in the mechanism of failed myocardium repair after stem cells transplantation.

The effects of DNA methylation inhibitor 5-azacytidine (5-aza-C) and histone acetylation inhibitor trichostatine A (TSA) on the structure of pericentric heterochromatin in cultured mouse cells (L929) has been studied. After 48 h of 5-aza-C treatment, about 85% of the cells demonstrate transformation of chromocenters from ovoid to elongated structures. Hypotonic treatment of these cells reveals tandemly arranged DAPI-positive globules, well distinguishable by light microscopy. The same globular units can be revealed in hypotonic-treated control cells. 48 h of TSA treatment causes dramatic decrease in HP 1alpha content in the cells. Chromocenters in 25% of treated cells became highly decondenced and can not be reliably detected by light and electron microscopy. 85% of cells demonstrate globular chromocenters with low HP 1alpha content. Hypotonic treatment causes transformation of compact chromocenters into ring-like structures, which can be either single or clustered. Rings are formed by uniform fiber, in which no globular subunits are detected. The data obtained are discussed concerning several mechanisms of heterochromatin structure maintenance and the roles of epigenetic marks in them.

Conformational changes of in chromatin structure play a key role in the regulation of intranuclear processes and, therefore, are under advanced study. In the paper presented, the fine structure of chromatin in DNA replication sites was examined in cells fixed in situ and in cells permeabilized in low ionic strength solutions in the presence of divalent cations. The method provides the visualization of higher-level chromatin structures, globular chromomeres, and chromonema fibres. Nascent DNA was detected on the surface of ultrathin sections immunochemically using anti-BrdU antibodies. It was shown that newly replicated DNA preferentially localizes within the zones filled with globular and fibrillar elements 30 nm in diameter. DNA-completed replication became embedded in 60–100-nm-thick chromonema elements. The results are discussed in the context of the hierarchical folding of chromatin fibers.