Chromatin Migration Research Articles

Особенности мужского мейоза на примере четырех сортов овса посевного (Avena sativa L.), возделываемых в условиях Центральной Якутии

Extreme environmental factors decrease seed setting and form unseeded or thinning panicles. Since meiosis underlies formation of gametes, it largely determines further processes associated with pollination, fertilization and development of a full-fledged seed. In this regard, studies meant to identify the peculiarities of male meiosis for plants cultivated in the severe natural and climatic conditions of Central Yakutia are relevant and of particular interest. The goal of this study was to identify the characteristics of male meiosis in oat (Avena sativa L.) varieties adapted to local conditions. We should emphasize that no studies of meiosis in common oats under the conditions of Central Yakutia have been performed earlier. We used four varieties of common oats (Pokrovsky, Vilensky, Pokrovsky 9, and Khibiny 2) as study material. All the varieties under analysis were hexaploids (2n = 6x = 42). The studies were conducted in 2020 at the Pokrovsky Department of Yakut Research Institute of Agriculture, in Khangalassky ulus of the Republic of Sakha (Yakutia) located in the middle taiga zone. The weather and climatic conditions during the growing season were characterized by an insufficient provision of soil with productive moisture at the early phases of grain and forage crop growth and development. To analyze meiosis, we used oat spikelets sampled when they were emerging into the tube. The spikelets were fixed on June 25-27, 2020 in duplicate using Carnoy’s solution. We analyzed microsporogenesis and pollen fertility on the temporary squashed preparations of meiocytes using the generally accepted method with 4% acetocarmine staining. The results were statistically processed using Statistica 7.0 (StatSoft. Inc., USA). The samples were compared through one-way ANOVA. The general cytological picture of meiosis in the oat varieties under study mostly corresponded to the conservative scenario typical of monocotyledonous plants. However, all the oat varieties showed some deviations from the general meiosis pattern. One of such deviations was cytomixis - chromatin migration as part of nuclei between microsporocytes through one or several cytomictic channels (Fig. 1, 2). As a rule, the phenomenon of cytomixis in microsporocytes was observed at middle prophase 1 at the pachytene stage (Fig. 1). Nuclei migration was also observed at late prophase 1, for example, at the diplotene stage. Chromatin movement between two adjacent microsporocytes clearly correlated with the formation of micronuclei and cytoplasts. Quite often, this process was also accompanied by nuclei pycnosis (Fig. 2). We should note that rarely we observed massive destructive cytomixis in microsporocytes and tapetum cells. When studying common oat cultivars, we also found microsporocytes with separately lying bivalents that were not included in the metaphase plate (Fig. 2), as well as chromosomal bridges (Fig. 2) at the anaphase-telophase 1 stage. The proportion of microsporocytes with meiotic deviations in all the four cultivars was small (Fig. 3) and varied from 2.99 to 3.23% (Table 1). We found no consequences of the revealed deviations at the tetrad stage. Probably, either most deviations were compensated for by intracellular mechanisms or defective microsporocytes were excluded from the microsporogenesis and eliminated. Besides, these deviations of meiosis did not significantly decrease pollen fertility (Table 1). The results indicate that the observed deviations of meiosis do not significantly contribute to seed formation in such an important grain fodder crop as common oats in the climatic conditions of Central Yakutia. The article contains 3 Figures, 2 Tables, 27References. The Authors declare no conflict of interest.

Cytomixis in angiosperms from Northwestern Argentina

ABSTRACT Cytomixis consists of the migration of chromatin and/or organelles between proximate cells through intercellular channels. Cytomixis has been detected in 10 taxa belonging to seven families from northwestern Argentina (NWA). The aim of this work was to report the presence of cytomixis in different species, genera and families of angiosperms from NWA. Plant material was collected from different localities. Floral buds were fixed and stained using classical cytogenetic techniques. Cytomixis was observed in both meiotic divisions and was characterized by the connection and passage of substance between two or more cells. Meiotic irregularities, such as agglutinated chromosomes, lagging chromosomes, the formation of bridges, micronuclei, pyknosis were observed in the studied taxa which resulted into the formation of monads, dyads, pentads, and hexads during microsporogenesis. In one of the species, Piper hieronymi, formation of unreduced 2 n pollen grains was also recorded. In this paper, we also provided the lists of 84 families of angiosperms where the phenomenon of cytomixis has been reported so far in the literature. The current paper adds some families and genera where the phenomenon of cytomixis has been reported for the first time.

First detection of cytomixis and its consequences in Thalictrum cultratum Wall. (Ranunculaceae)

In current paper, we studied the meiotic chromosome number and details of cytomixis recorded for the first time in Thalictrum cultratum Wall. from the high altitude areas of northwest Himalaya (India). Cytomixis, the phenomenon of inter PMC (pollen mother cell) migration of chromatin material has been reported in many angiosperms and other groups of plants, but there is no published report in Thalictrum cultratum. All the presently studied accessions of the species existed at diploid level (x = 7). The present chromosome count of n = 14 has been reported for the first time from the study area. The cytological stability of any plant is an important consideration in view of its existence in nature. Meiotic disturbances can impose threat to the reproductive potential of plant. Current study reveals the occurrence of inter PMC migration of chromatin material. In present investigation, the phenomenon of cytomixis can be observed between 2 to 9 PMCs. Chromatin migration occurs through narrow and broad cytoplasmic channels or occasionally fused together during metaphase-I and anaphase-I. Cytomixis resulted in the formation of hypo- (n = 2, 2 + 1 small fragment, n = 3 + 3 small fragments, 4, 5 + extra mass of chromatin, 13) and hyperploid (n = 7 + 2 small fragments, 8 + 2 small fragments, 14, 14 + 2 small fragments, 15) PMCs, variable sized pollen grains and pollen sterility (20%). The current study reveals that cytomixis is responsible for formation of PMCs with variable chromosomes (hypoand hyperploid) and pollen grains of heterogeneous sizes and pollen sterility in T. cultratum. This is the first report of cytomixis in the species.

Microscopic Observation, Three-dimensional Reconstruction, and Volume Measurements of Sperm Nuclei

Karyogamy, a migration of the sperm nucleus toward the egg nucleus and their subsequent nuclear fusion, is an important biological event for initiating zygote formation toward early embryogenesis in angiosperms. However, how the male nucleus approaches and fuses with the female nucleus still remains unclear. Recently, time-lapse measurement of nuclear volume during karyogamic events revealed that the sperm nucleus enlarges during contact with the egg nucleus via possible one-directional migration of egg chromatin into sperm nucleus (Ohnishi et al., 2014). Here, we describe the protocol for microscopic observation, three-dimensional reconstruction, and volume measurements of sperm nuclei in rice zygotes/fused gametes, which are produced by an in vitro fertilization system (Uchiumi et al., 2006; Uchiumi et al., 2007). The present protocol will be applied for monitoring nuclear dynamics in cells during cell division, differentiation, de-differentiation and polarity formation as well as karyogamy progression.

Bleomycin-induced γH2AX foci map preferentially to replicating domains in CHO9 interphase nuclei

Exposure to DNA damaging agents triggers phosphorylation of histone variant H2AX (generating γH2AX) in large chromatin regions flanking DNA lesions, allowing their immunodetection as nuclear foci. Even though a predominance of γH2AX foci in euchromatin has been postulated, foci positioning when DNA insult occurs in replicating eu- or heterochromatin regions has not been extensively explored. Labeling of interphase nuclei with 5-ethynyl-2'-deoxyuridine (EdU) pulses has revealed that DNA replication is temporarily and spatially regulated: euchromatin replicates in early S (ES) and heterochromatin along mid and late S (MS/LS) phases. In order to map DNA damage with respect to replicating domains, the distribution of γH2AX foci induced by the radiomimetic agent bleomycin was analyzed in CHO9 interphase nuclei by delineating euchromatic (H3K4me3+) and replicating (EdU+) regions. Quantification of overlapping pixels and 3D inter-object overlap in binary masks revealed colocalization between γH2AX foci and EdU + domains both in ES and MS/LS nuclei, indicating that primary damage distribution is modulated by DNA synthesis. Further, we verified that EdU incorporation by itself did not influence BLEO-induced γH2AX nuclear patterns. Our results also revealed a repeated localization of γH2AX foci in replicating/nonreplicating interfaces which could reflect short-range chromatin migration following DNA insult.

First Report of Cytomixis and Meiotic Abnormalities in <i>Nepeta govaniana</i> from Solang Valley, Kullu District, Himachal Pradesh

In the present study, we report the phenomenon of cytomixis for the first time in some individuals of Nepeta govaniana showing chromatin transfer at the different stages of meiosis I. In most of the cases, two to six PMCs are involved in chromatin transfer. The migration of chromatin is either complete or partial, resulting in denucleated, double nucleolated, hypoploid and hyperploid PMCs. Single or multiple cytoplasmic channels between two or more proximate PMCs have been observed at prophase I. PMCs involved in cytomixis showed various other meiotic irregularities such as spindle abnormalities, chromatin stickiness, asynchronous disjunction of bivalents and laggards. In the present case, the cytomixis and associated meiotic irregularities did not cause significant reduction in pollen fertility, but resulted into the formation of heterogeneously sized pollen grains. The role of such variably sized fertile pollen grains in the origin of polyploids and aneuploids can not be ruled out.

EMS induced intercellular chromatin transmigration in Papaver somniferum L.

The phenomenon of chromatin migration was observed during microsporogenesis in an ethyl methane sulphonate (EMS) treated population of poppy, which is an important medicinal plant. Cytomixis occurred through a cytoplasmic channel or by direct fusion of pollen mother cells (PMCs); the former was more recurring than the latter. The process was associated with irregular meiosis. PMCs with differing chromosome numbers from the normal diploid number (2n = 22) through cytomixis may lead to the production of aneuploid and polyploid gametes. An increase in the concentration of EMS had a positive effect on the percentage of PMCs showing cytomixis. In addition to cytomixis, other chromosomal abnormalities were also found. Cytomixis along with the related chromosomal abnormalities largely affected the post-meiotic products resulting in some pollen sterility.

An ultrastructural study of microsporogenesis in tobacco line SR1

This article provides an ultrastructural atlas of microsporogenesis in the tobacco model line SR1. The stages of cell-wall remodeling and reorganization of the intercellular channels, accompanying this process, are reported for the microspore mother cells. The meiotic changes in the cell nucleus and cytoplasm are traced. The appearance of single-, double-, or multi-membrane nuclear vacuoles in microspore mother cells and their further elimination from the nucleus are for the first time described for the genus Nicotiana as well as deviations from a normal course for this process. Intercellular chromatin migration (cytomixis) was observed in the microsporogenesis of the line SR1 and behavior of the nuclear vacuoles within the cytomictic nucleus was described for the first time. The enzymatic activity of spherosome-like vesicles in the tobacco microsporogenesis is discussed. The features of microsporogenesis in the tobacco line SR1 are compared with those of other plant species and its association with the transition from a diploid to a haploid phase of the life cycle is discussed.

EMS-induced cytomictic variability in safflower (Carthamus tinctorius L.)

Seeds of safflower (Carthamus tinctorius L.) were subjected to three treatment durations (3h, 5h and 7h) of 0.5 % Ethyl Methane Sulphonate (EMS). Microsporogenesis was carried out in the control as well as in the treated materials. EMS treated plants showed interesting feature of partial inter-meiocyte chromatin migration through channel formation, beak formation or direct cell fusion. Another interesting feature noticed during the study was the fusion among tetrads due to wall dissolution. The phenomenon of cytomixis was recorded at nearly all the stages of microsporogenesis connecting from a few to several meiocytes. Other abnormalities such as laggards, precocious movement, bridge and non-disjunction of chromosomes were also recorded but in very low frequencies. The phenomenon of cytomixis increased along with the increase in treatment duration of EMS. Cells with these types of cytomictic disturbances may probably result in uneven formation of gametes or zygote, heterogenous sized pollen grains or even loss of fertility in future.

An ultrastructural study of cytomixis in tobacco pollen mother cells

Intercellular chromatin migration (cytomixis) in the pollen mother cells of two tobacco (Nicotiana tabacum L.) lines was analyzed by electron microscopy during the first meiotic prophase. The maximal manifestation of cytomixis was observed in the pachytene. As a rule, several cells connected with one another by cytomictic channels wherein the nuclei migrated were observable at this stage. In the majority of cases, nuclei passed from cell to cell concurrently through several closely located cytomictic channels. Chromatin migrated between cells within the nuclear envelope, and its disintegration was unobservable. The nucleus, after passing through cytomictic channels into another cell, can be divided into individual micronuclei or, in the case of a direct contact with another nucleus, can form a nuclear bridge. It has been demonstrated that the chromatin structure after intracellular migration visually matches the chromatin structure before it passed through the cytomictic channel. No signs of pyknosis were observable in the chromatin of the micronuclei formed after cytomixis, and the synaptonemal complex was distinctly seen. The dynamics of changes in the nucleoli during cytomixis was for the first time monitored on an ultrastructural level. Possible mechanisms determining cytomixis are discussed and the significance of this process in plant development is considered.

Effect of Cytomixis on the Pollen Size in 'Seabuckthorn' (Hippophae rhamnoides L., Elaeagnaceae)

Present study reveals the occurrence of inter PMC transfer of chromatin material in the PMCs of ‘Seabuckthorn’ (Hippophae rhamnoides L.). The migration of chromatin is observed from early prophase up to telophase-I. In majority of the cases 2–4 PMCs are involved in chromatin transfer, however in rare occasions migration of chromatin occurs in groups. The migration of chromatin is observed to be partial as well as complete, which resulted into the formation of anucleated, hypo and hyperploid PMCs. In rare cases, direct fusion between two PMCs at AI lead to the formation of a polyploid PMC with 36 chromosomes. As a consequence of cytomixis various other associated meiotic anomalies such as chromatin stickiness at MI and late disjunction of bivalents at AI, pycnotic chromatin, bridges and laggards at anaphases and telophases are resulted. Microsporogenesis is also abnormal resulting in the formation of dyads, triads, and polyads with micronuclei. The cytomixis though do not cause much pollen sterility but it surely results in the formation of heterogenous sized fertile pollen grains. The pollen grains are classified into small, medium and large sized with medium sized pollen grains being in high frequency. The role of such fertile, especially, small and large sized pollen grains, in the origin of plants with aneuploid and polyploid chromosome number, can not be ruled out. The present meiotic count of n=9 is the first record for the study area.

Cytology of Caltha palustris L. (Ranunculaceae) from Cold Regions of Western Himalayas

Caltha palustris L. (Ranunculaceae), popularly known as Kingcup or Marsh Marigold is a polymorphic species distributed in the cold temperate regions of western Himalayas. The species exhibits considerable amount of intraspecific morphological and chromosomal diversity involving polyploidy, aneuploidy and hybridization. Besides, some individuals also show the presence of B-chromosomes. Presently three populations from the cold desert region of Kullu and Lahaul-Spiti districts of Himachal Pradesh have been studied for detailed meiotic studies. These populations which uniformly show the presence of 16 bivalents at M-I are tetraploid on x=8. Presence of eight large sized and eight small sized bivalents at M-I, suggest these populations to be allotetraploid. Majority of the individuals in one of the populations show normal meiotic course and high pollen fertility. However, in the other two populations, the individuals show inter PMC transfer of chromatin material at various stages of meiosis and associated meiotic irregularities such as chromosome stickiness, pycnotic chromatin, late disjunction of bivalents, micronuclei and chromatin bridges at anaphases and telophases. Besides, the chromatin migration also results in the formation of aneuploid with 8, 11 and 13 bivalents and anucleated PMCs. Although the phenomenon of cytomixis does not affect apparent pollen fertility but it surely resulted into heterogenous sized pollen grains. Although the genetic constitution and fate of such apparently fertile heterogenous sized pollen grains is yet to be ascertained, but their possible role in the development of individuals with varied chromosome number could not be ruled out. The phenomenon of chromatin transfer in the species seems to be under direct genetic control as the individuals with and without cytomixis found to grow in the same environmental conditions.

Cytomixis in pollen mother cells of Medicago sativa L.

Cytomixis (i.e., chromatin migration between meiocytes) has been detected in many plant species, but not in Medicago sativa spp. In the present study we report the identification of a few cytomictic alfalfa plants. Those plants, the "mother plants," were selfed and crossed with a normal control plant. Microsporogenesis analysis was performed on the mother plants, on the S(1) and F(1) plants, and on controls. The S(1) and F(1) plants, like the mother plants, were found to be cytomictic. Single or multiple chromatin bridges between two or more meiocytes were observed almost exclusively in prophase I. Some completely empty meiocytes were also observed. In addition to cytomixis, other meiotic abnormalities were found. Control plants showed an almost regular meiosis. The highest values of cytomixis were observed in the mother plants, and the lowest in their F(1) progenies. Variability of cytomixis in the F(1) plants is probably due to a heterozygotic condition of the parents for this trait. No significant correlation was found between cytomixis and pollen viability, even if the cytomictic plants showed low values of pollen viability.

The arrangement of microtrabecular network during chromatin migration process and immunolocalization of actin and myosin in pollen mother cells of David Lily

Abstract The pollen mother cells of David lily at synizesis of meiotic prophase I were studied using the light microscope and diethylene glycol distearate (DGD) embedment-free electron microscope. The results showed that there existed clear areas in front of and behind the chromatin masses undergoing migration via cytoplasmic channels (cytomixis) under light microscope. Observations of diethylene glycol distearate embedment free electron microscopy indicated that these clear areas were actually filled with microtrabecular components. Several major arrangement changes of microtrabecular network were observed as follows: When the nucleus moved aside from the center, an area filled with cytoplasmic microtrabecular network appeared behind. Later, when chromatin underwent cytomixis via cytoplasmic channels, another area filled with nuclear microtrabecular network appeared between nuclear envelope and the rear condensed chromatin, and some thick microtrabecular filaments linked the migrating chromatin masses in...

Effects of ethidium bromide on DNA loop organisation in human lymphocytes measured by anomalous viscosity time dependence and single cell gel electrophoresis

The effects of ethidium bromide (EtBr) on human lymphocytes were studied by the method of anomalous viscosity time dependence (AVTD) and by the comet assay. EtBr at low concentrations increased the maximum viscosity and time of radial migration as measured with AVTD at neutral conditions of lysis. A pronounced relaxation of DNA loops was observed with the neutral comet assay. The maximal comet length corresponded to 2 Mb DNA loops. At high concentrations of EtBr, 2 mg/ml, significant reduction in AVTD below control level was seen that suggested hypercondensation of chromatin. The hypercondensation was directly observed with the neutral comet assay. EtBr did not induce DNA strand breaks as measured by the alkaline comet assay. The hypercondensed nuclei could be decondensed by irradiation with γ-rays or exposure to light. The data provide evidence that EtBr at high concentrations resulted in hypercondensation of chromatin below control level. The comet assay confirmed that the increase in AVTD peaks deals with relaxation of loops and AVTD decrease is caused by chromatin condensation. The prediction of the AVTD theory for a correlation between time of radial migration and condensation of chromatin was verified. Further, the data show that the comet assay at neutral conditions of lysis is rather sensitive to DNA loop relaxation in the absence of DNA damage. Finally, donor specificity was found for the hypercondensation.

In vitro dynamics of chromatin organization and migration.

The organization of eukaryotic chromatin is not static but changes as a function of cell status during processes such as proliferation, differentiation, and migration. DNA quantification has not been used extensively to investigate chromatin dynamics in combination with cellular migration. In this context, an optimized DNA-specific, nonperturbant method has been developed for studying chromatin organization, using the fluorescent vital bisbenzimidazole probe Hoechst 33342: this property has been described by Hamori et al. (1980). Computer-assisted image analysis was used to follow migratory activity and chromatin organization of L929 fibroblasts during in vitro wound healing. Cell movements were analyzed using an optical flow technique, which consists in the calculation of the velocity field of cells and nuclear movements in the frame. This system allows the correlation of cell migration and position in the cell cycle. It makes it possible to study chromatin dynamics using a quantitative analysis of nuclear differentiation reorganization (nuclear texture) and to correlate this with migration characteristics. The present system would be of interest for studying cell-extracellular matrix interactions using differing substrates, and also the migratory response to chemotactic factors. Such a model is a prerequisite for gaining better understanding of drug action.

Spontaneous Cytomixis in the Microsporogenesis of Sweet Pea Lathyrus odoratus L. (Leguminosae).

Cytomixis is described as an spontaneous phenomenon in L. odoratus. Cytoplasmic channels were seen at all stages of microsporogenesis connecting from few to several meiocytes. Chromatin migration between cells was observed mainly at early prophase, metaphase II and telophase II with decreasing frequencies as meiosis advanced. Cytological analysis revealed other abnormalities in very low frequencies such as alteration of bivalents disjuction, laggards and bridges. Degenerating gametes as well as nonreduced gametes and microgametes were observed, although their frequencies varied depending on anthers and floral buds. Significance of cytomixis in pollen viability and seed set is discussed.

Formation of secondary plasmodesmata and intercellular passages by means of ACHT

By artificially forcing cytoplasm and chromatin migration between different plant cells and through coculture, the isolation layer with uneven thickness was formed between tobacco and spinach cells. Then it was gradually absorbed, thinned, and eliminated finally, so that different plant cells were linked together with each other into chimaera. Under further co-culture condition, secondary half-plasmodesmata, secondary plasmodesmata and intercellular passages (cytomictic channels) were formed on one side or both sides of the walls of the adjoining cells; thus different plant cells were linked into symplastic cell mass. This provides the cell structural and cell physiological basis for further manipulating the intercellular migration of cytoplasm and chromatin. Various intercellular contacts and passages are described.

STUDY ON WAYS OF FORCING INTERCELLULAR CYTOPLASM AND CHROMATIN MIGRATION TO TRANSFER EXTERNAL GENETIC SUBSTANCES TO PLANT CELLS BY ARTIFICIAL MOTIVE FORCE

A new cell engineering technique (L. B. technique) was established in our Lab. At first, the physical and chemical methods were used to facilitate the reestablishment of intercellular contacts and plasmodesma channels between different parents, thus forcing cytoplasm and chromatin to pass the cell wall with different qualities and quantities from one cell to others, through the enlarged intercellular plasmodesma channels or the vulnerable regions and the holes on the cell wall formed differently in growth and thickness in the process of cell wall formation to introduce external genetic substances or gene groups into plant cells. There are different ways, frequencies and strengths for the migration between the cells in different growth and development regions or the same growth and development region. In this paper we advance the mechanism of cytoplasm and chromatin migration through the cell wall: There are a large number of plasmodesma channels or vulnerable regions and holes different in growth and thickness during the formation of the cell walls between meristematic cells. Low osmotic pressure and low pH value K+ solution treatments force the augmentation of plasmic streaming, and the streaming of karyolymph, the relaxation of the cell wall, the formation of more holes on the cell wall and the enlargement of plasmodesma channels in meristematic cells. All these make a proper structural and physiological condition for migrating through cell walls. Osmotic pressure and centrifugal forces are the practical forces forcing cytoplasm and chromatin to migrate extensively and transfer external genetic substances or gene groups to plant cells.

Cytomixis in woody species

Cytomixis with actual transfer of chromatin material has been recorded in 6 woody species, from early prophase to telophase-II. It is more common at early stages of meiosis. Number of pollen mother cells involved vary from 2–8. As a consequence of chromatin migration both hypo and hyperploid meiocytes are seen inSerissa foetida, Symplocos chinensis andQuercus semecarpifolia. However, incordia dichotoma andSalix elegans lower and inCaryopteris odorata higher numbers than the normal complement are not countable due to stickiness or agglutination of chromosomes, respectively. Reduction in pollen fertility in these species is due to the cytomixis. The phenomenon is attributed to certain unknown genetic factors.