Chromatin Masses Research Articles

The paternal sex ratio chromosome in the parasitic wasp Trichogramma kaykai condenses the paternal chromosomes into a dense chromatin mass.

A recently discovered B chromosome in the parasitoid wasp Trichogramma kaykai was found to be transmitted through males only. Shortly after fertilization, this chromosome eliminates the paternal chromosome set leaving the maternal chromosomes and itself intact. Consequently, the sex ratio in these wasps is changed in favour of males by modifying fertilized diploid eggs into male haploid offspring. In this study, we show that in fertilized eggs at the first mitosis the paternal sex ratio (PSR) chromosome condenses the paternal chromosomes into a so-called paternal chromatin mass (PCM). During this process, the PSR chromosome is morphologically unaffected and is incorporated into the nucleus containing the maternal chromosomes. In the first five mitotic divisions, 67% of the PCMs are associated with one of the nuclei in the embryo. Furthermore, in embryos with an unassociated PCM, all nuclei are at the same mitotic stage, whereas 68% of the PCM-associated nuclei are at a different mitotic phase than the other nuclei in the embryo. Our observations reveal an obvious similarity of the mode of action of the PSR chromosome in T. kaykai with that of the PSR-induced paternal genome loss in the unrelated wasp Nasonia vitripennis.

In vivo characterization of regulatory polymorphisms by allele-specific quantification of RNA polymerase loading.

In vivo characterization of regulatory polymorphisms is a key requirement for next-generation human genetic analysis. Here we describe haploChIP, a method that uses chromatin immunoprecipitation (ChIP) and mass spectrometry to identify differential protein-DNA binding in vivo associated with allelic variants of a gene. We demonstrate this approach with the imprinted gene SNRPN. HaploChIP showed close correlation between the level of bound phosphorylated RNA polymerase II at the SNRPN locus and allele-specific expression. Application of the approach to the TNF/LTA locus identified functionally important haplotypes that correlate with allele-specific transcription of LTA. The haploChIP method may be useful in high-throughput screening for common DNA polymorphisms that affect gene regulation in vivo.

Highly Unstable Male Meiosis in Two Aberrant Plants Isolated from the Mutant Population of Nigella damascena L. (love-in-a-mist)

Two aberrant (plant type I and II) plants possessing meiotic instability in the microsporocytes were identified from the mutant population of Nigella damascena L. (commonly known as love-in-a-mist; family: Ranunculaceae). Plant type I (screened from 10 kR M1 generation) was a weak plant with small sized flowers and shrunken anthers devoid of any pollen grains at anthesis. The plant dried up at blooming stage. Male meiotic study revealed the occurrence of condensed and compact chromatin masses of variable sizes and numbers (12 to 13 mostly and 3 to 5 rarely) and occasional formation of bivalent and univalent like configurations in the meiocytes. Plant type II (isolated at M2 from the M1 progeny of 0.25% EMS treatment) was a small flower mutant (SF2), the morphological nature and chromosomal behaviour of which have been compared to control and SF1 (small flower mutant arising in different M2 line). SF1 showed similar morphological behaviour as SF2 but demonstrated normal meiosis like control. SF2 manifested hypoploid (2n 12) chromosome numbers at MI and AI and prevalence of unstable meiosis in the form of asynaptic and desynaptic chromosome behaviour, differential condensation of chromosomes, persistent nucleolar body and occasional multivalent formation in first and mid-flowering phases. SF2 showed more diploid PMCs at late flowering phase. SF2 was also found to be associated with defects in female gametophytic tissues. Possible mode of gene action in the plant types has been predicted.

The ABCs of SMC proteins: two-armed ATPases for chromosome condensation, cohesion, and repair.

The first draft of the human genome sequence was reported a year ago. It may be a good time to remind ourselves that the genetic information encoded in the ∼3000-Mb sequence is stored not only in the public or private databases but also in the tiny space of the cell nucleus. The total length of human genomic DNA, which resides in 23 chromosomes, reaches approximately one meter. It is by no means a simple task to fold up the long DNA molecules and package them within a cell nucleus whose diameter is only ∼10 μm. Even more striking is that the DNA molecules are faithfully duplicated and segregated into two daughter cells in an extremely limited space. Although more than 100 years have passed since Walther Flemming first described the dynamic behavior of chromosomes (or mitosis) during cell division, it remains highly mysterious how this remarkable process of chromosome segregation is achieved at a mechanistic level. From a cytological point of view, two dramatic events occur on chromosomes during mitosis. The first one is the conversion of an amorphous mass of interphase chromatin into a discrete set of rod-shaped chromosomes (chromosome condensation), which occurs from prophase to metaphase (Koshland and Strunnikov 1996; Hirano 2000). The second is the splitting of chromosomes into two halves, which takes place highly synchronously at the onset of anaphase (Dej and Orr-Weaver 2000; Nasmyth et al. 2000). As a crucial prerequisite for these events, duplicated chromosomes (sister chromatids) must be held together immediately after DNA replication in S phase and throughout G2 phase. The importance of this process (sister chromatid cohesion) has been fully appreciated only recently because the pairing of sister chromatids cannot be visualized by classical cytology before chromosomes condense in early mitosis. Recent genetic and biochemical studies have begun to shed light on the molecular mechanisms underlying cohesion, condensation, and separation of chromosomes during the mitotic cell cycle. One of the unexpected findings is that chromosome condensation and sister chromatid cohesion are regulated by distinct, yet structurally similar, protein complexes termed condensin and cohesin, respectively. At the heart of the two protein complexes lie members of a family of chromosomal ATPases, the structural maintenance of chromosomes (SMC) family. Equally intriguing, SMC proteins are found in most, if not all, bacterial and archaeal species, implicating that their fundamental contribution to chromosome dynamics started even before the acquisition of histones during evolution. The goal of this review article is to discuss the current understanding of higher-order chromosome dynamics with an emphasis on the role of SMC proteins. I start with the basic description and classification of SMC proteins and then summarize emerging information on the diverse chromosomal functions supported by SMC proteins. Finally, I discuss the mechanistic aspects of bacterial and eukaryotic SMC proteins and try to make an integrated picture of their seemingly different actions.

Why so many noncoding nucleotides? The eukaryote genome as an epigenetic machine.

It is recalled that dispensability of sequences and neutral substitution rate must not be construed to be markers of nonfunctionality. Different aspects of functionality relate to differently-sized nucleotide communities. At the time cells became nucleated, a boom of epigenetic processes led to uses of DNA that required many more nucleotides operating collectively than do functions definable in terms of classical genetics. Each order of magnitude of nucleotide plurality was colonized by functions germane to that order. The eukaryote genome became a great epigenetic machine. Sequences of different levels of nucleotide plurality are briefly discussed from the point of view of their functional relevance. By their activities as both transcribed genes and cis-acting repeats, SINEs and LINEs are the principal link between genetic and epigenetic processes. SINEs can act as local repeats to produce position effect variegation (PEV) in a nearby gene. PEV may thus represent a general method of overall transcriptional regulation at the level of cell collectivities. When tracking the scale dependence of nucleotide function, one finds the 100 kb order of nucleotide plurality to provide epigenetically the basis at once for PEV, imprinting, and cell determination, with sectorial repressibility a trait common to the three. In sectorial repressibility, introns may play a structural role favoring the stability of higher-order chromatin structures. At that level of nucleotide involvement, nonconserved nonhomologous nonprotein-coding sequences may often play the same structural roles. In addition, genomic distance per se--and, therefore, the mass of intervening nucleotides--can have functional effects. Distances between enhancers and promoters need to be probed in this respect. At the 1,000 kb level of nucleotide function, attention is focused on the formation of centromeres. It is one of the levels of nucleotide plurality per function where specificity in the generation of DNA/protein complexes seems to depend more upon the structural fit among factors than upon the DNA sequence. This circumstance may explain in part the prevailing difficulty in recognizing the functional nature of sequences among non-protein-coding nucleotide arrays and the propensity among investigators to tag the majority of DNA sequences in higher organisms as functionally meaningless. Noncoding DNA often may not be 'selected' as an appropriate niche for a certain function, but be 'elected' in that capacity by a group of factors, as a preexisting sequence that is only now called upon to serve. Much of the non-protein-coding DNA may thus be only conditionally functional and in fact may never be elected to functions at a high level of nucleotide plurality. Eukaryotes are composites, at different levels of this plurality, of the functional and the nonfunctional, as well as of the conditionally functional and the outright functional. Thus, a sequence that is nonfunctional at one level of nucleotide plurality may participate in a functional sequence at a more inclusive level. In the end, every nucleotide is at least infinitesimally functional if, for metabolic and developmental reasons, the chromatin mass as such becomes a selectable entity. Given the scale dependence of nucleotide function, large amounts of 'junk DNA', contrary to common belief, must be assumed to contribute to the complexity of gene interaction systems and of organisms.

Ultrastructural staining with sodium metaperiodate and sodium borohydride.

This article describes new ultrastructural staining methods for osmicated tissues based on the incubation of sections with sodium metaperiodate and sodium borohydride solutions before uranyl/lead staining. Sections incubated with sodium metaperiodate and sodium borohydride, treated with Triton X-100, and stained with ethanolic uranyl acetate/lead citrate showed a good contrast for the nucleolus and the interchromatin region, whereas the chromatin masses were bleached. Chromatin bleaching depended on the incubation with these oxidizing (metaperiodate) and reducing (borohydride) agents. Other factors that influenced the staining of the chromatin masses were the en bloc staining with uranyl acetate, the incubation of sections with Triton X-100, and the staining with aqueous or ethanolic uranyl acetate. The combination of these factors on sections treated with metaperiodate/borohydride provided a different appearance to the chromatin, from bleached to highly contrasted. Most cytoplasmic organelles showed a similar appearance with these procedures than with conventional uranyl/lead staining. However, when sections were incubated with metaperiodate/borohydride and Triton X-100 before uranyl/lead staining, the collagen fibers, and the glycocalix and zymogen granules of pancreatic acinar cells, appeared bleached. The possible combination of these methods with the immunolocalization of the amino acid taurine was also analyzed. (J Histochem Cytochem 50:11-19, 2002)

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...

Cell death may regulate visual functionality in the retina of adults of the dipteran Ceratitis capitata

The white eye mutation in the medfly Ceratitis capitata, like the homologous mutation in Drosophila melanogaster, was shown to impair visual function. Light and electron microscopy, combined with the DNA-end labelling histochemistry (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) technique), were used to investigate whether programmed cell death may contribute to the morpho-functional differences between the retina of adults from the white eye and wild type strains. Several photoreceptor nuclei in mature white eye flies appeared smaller and showed intensely Toluidine Blue-stained chromatin masses. At the ultrastructural level, they showed different stages of degeneration, resembling apoptotic figures. Positive TUNEL labelling in the white eye retina indicates that apoptosis may be a candidate mechanism for retinal cell degeneration in adult flies, where visual functionality is altered, to achieve the proper cell number. Apoptosis also appears to occur in the wild type retina in early adult life during normal tissue development.

Programmed cell death in the pancreas of Bufo bufo during metamorphosis.

Programmed cell death or apoptosis occurred in anuran amphibian larval pancreas as a remodelling agent, and was responsible for the reduction of the gland volume during metamorphosis. Apoptotic cells were recognisable by their morphological characteristics and could be immunocytochemically detected by means of the TUNEL reaction, which evidenced nuclear DNA fragmentation. During the last stages of prometamorphosis, that is in the period of hindlimb differentiation, only a few TUNEL positive cells occurred, whereas they increased at the beginning of metamorphic climax, that is at forelimb emergence and during tail regression. Under the electron microscope, the typical morphological characteristics of apoptosis were observed: decrease in size, and the presence of wide intercellular spaces and nuclei with dense chromatin masses arranged in crescents. The fragmentation of these cells produced the so-called 'apoptotic bodies': portions of cytoplasm lined by a membrane, containing nuclear fragments and cytoplasmic organites. Dead cell elimination is hypothesised to occur by phagocytic ingestion.

Multiple myeloma: an odyssey of discovery

Multiple myeloma: an odyssey of discovery

Cathepsin D Deficiency Induces Lysosomal Storage with Ceroid Lipofuscin in Mouse CNS Neurons

Cathepsin D-deficient (CD-/-) mice have been shown to manifest seizures and become blind near the terminal stage [approximately postnatal day (P) 26]. We therefore examined the morphological, immunocytochemical, and biochemical features of CNS tissues of these mice. By electron microscopy, autophagosome/autolysosome-like bodies containing part of the cytoplasm, granular osmiophilic deposits, and fingerprint profiles were demonstrated in the neuronal perikarya of CD-/- mouse brains after P20. Autophagosomes and granular osmiophilic deposits were detected in neurons at P0 but were few in number, whereas they increased in the neuronal perikarya within days after birth. Some large-sized neurons having autophagosome/autolysosome-like bodies in the perikarya appeared in the CNS tissues, especially in the thalamic region and the cerebral cortex, at P17. These lysosomal bodies occupied the perikarya of almost all neurons in CD-/- mouse brains obtained from P23 until the terminal stage. Because these neurons exhibited autofluorescence, it was considered that ceroid lipofuscin may accumulate in lysosomal structures of CD-/- neurons. Subunit c of mitochondrial ATP synthase was found to accumulate in the lysosomes of neurons, although the activity of tripeptidyl peptidase-I significantly increased in the brain. Moreover, neurons near the terminal stage were often shrunken and possessed irregular nuclei through which small dense chromatin masses were scattered. These results suggest that the CNS neurons in CD-/- mice show a new form of lysosomal accumulation disease with a phenotype resembling neuronal ceroid lipofuscinosis.

RELEASE OF MITOTIC DESCENDANTS BY GIANT CELLS FROM IRRADIATED BURKITT'S LYMPHOMA CELL LINES

Polyploid giant cells are produced as part of the response of p53 mutant Burkitt's lymphoma cell lines to high doses of irradiation. Polyploid giant cells arise by endo-reduplication in the first week after a single 10 Gray dose of irradiation. Within the giant cells a sub-nuclear structure is apparent and within this, sub-nuclear autonomy is evident, as displayed by independent nuclear structure and DNA replication in different parts of the nucleus. The majority of these cells soon die as apoptotic polykaryons. However, approximately 10–20% of giant cells remain viable into the second week after irradiation and begin vigorous extrusion of large degraded chromatin masses. During the second week, the giant cells begin to reconstruct their nuclei into polyploid ‘bouquets’, where chromosome double-loops are formed. Subsequently, the bouquets return to an interphase state and separate into several secondary nuclei. The individual sub-nuclei then resume DNA synthesis with mitotic divisions and sequester cytoplasmic territories around themselves, giving rise to the secondary cells, which continue mitotic propagation. This process of giant cell formation, reorganization and breakdown appears to provide an additional mechanism for repairing double-strand DNA breaks within tumour cells.

300. Activated lymphocytes in manic-depressive patients

The state of the chromatin, i.e. compact or decondensed, is directly related to genomic expression. In view of reports suggesting immunological abnormalities in patients suffering from affective disorders, we designed this study to investigate ultrastructural conformation changes in chromatin, that would indicate the state of activation of the lymphocytes. The clinical sample consisted of 14 manic-depressive patients and 7 controls. The patients’ mean age was 42.2 years (age range 23–60). All patients fulfilled DSM-IV diagnostic criteria for bipolar affective disorder and had given informed consent to participate in the study. Blood was drawn by venipuncture and processed for electron microscopy. The leukocyte pellets were stained for histones with phosphotungstic acid hematoxylin (PTAH) and embedded in epoxy resins. The staining pattern of nuclei, in the manic-depressive group, was characterized by an increased staining density, implying decondensation of chromatin and an irregular distribution of the compact chromatin masses, indicating variably activated lymphocytes. Furthermore, the irregular outline of nuclei and the multiple nucleoli, are supporting the activated state of the lymphocytes. Additional markers of activation are the sub-membrane bands of endoplasmic reticulum, the villi-like projections of the cellular surface, the increased number of mitochondria and the frequent encounter of centrioles. The results of the study indicate that the lymphocytes of the patients are in a variably activated state, which is clearly and selectively made evident by the application of the PTAH staining method. These findings support the view that an immunological dysregulation is present in manic-depressive patients.

Neuronal type 1 apoptosis after unilateral focal ischemia with reperfusion in the P7 neonatal rat

Apoptotic cell death occurs frequently after cerebral ischemia, although prevailing necrosis could also be observed in older animals. The mechanisms that lead to delayed cell death after hypoxic-ischemic injury in the developing brain are not yet known. With silver staining, we showed increased dying neurons after ischemia and 4 to 24 hours of reperfusion in 7-day-old rats. Using electron microscopic studies, we investigated the presence of chromatin condensation. Injured neurons exhibited 2 different morphological types of apoptosis: Type 1 (aggregation of dense masses of chromatin beneath the intact nuclear membrane) predominately, and Type 2 (cytoplasmic vacuolization). Electron microscopy analysis also showed that the fragmented chromatin and neuronal debris were phagocytosed by surrounding glial cells, and associated with an inflammatory reaction leading to leukocyte infiltration. These results suggest that the immature brain may be more prone to apoptotic death with neuronal loss by neighboring cells. This experimental model may be useful for identifying the biochemical mechanisms that initiate and mediate Type 1 neuronal apoptosis after ischemia and reperfusion in neonate rodents Apoptotic cell death occurs frequently after cerebral ischemia, although prevailing necrosis could also be observed in older animals. The mechanisms that lead to delayed cell death after hypoxic-ischemic injury in the developing brain are not yet known. With silver staining, we showed increased dying neurons after ischemia and 4 to 24 hours of reperfusion in 7-day-old rats. Using electron microscopic studies, we investigated the presence of chromatin condensation. Injured neurons exhibited 2 different morphological types of apoptosis: Type 1 (aggregation of dense masses of chromatin beneath the intact nuclear membrane) predominately, and Type 2 (cytoplasmic vacuolization). Electron microscopy analysis also showed that the fragmented chromatin and neuronal debris were phagocytosed by surrounding glial cells, and associated with an inflammatory reaction leading to leukocyte infiltration. These results suggest that the immature brain may be more prone to apoptotic death with neuronal loss by neighboring cells. This experimental model may be useful for identifying the biochemical mechanisms that initiate and mediate Type 1 neuronal apoptosis after ischemia and reperfusion in neonate rodents

Anucleate Caenorhabditis elegans sperm can crawl, fertilize oocytes and direct anterior-posterior polarization of the 1-cell embryo.

It has long been appreciated that spermiogenesis, the cellular transformation of sessile spermatids into motile spermatozoa, occurs in the absence of new DNA transcription. However, few studies have addressed whether the physical presence of a sperm nucleus is required either during spermiogenesis or for subsequent sperm functions during egg activation and early zygotic development. To determine the role of the sperm nucleus in these processes, we analyzed two C. elegans mutants whose spermatids lack DNA. Here we show that these anucleate sperm not only differentiate into mature functional spermatozoa, but they also crawl toward and fertilize oocytes. Furthermore, we show that these anucleate sperm induce both normal egg activation and anterior-posterior polarity in the 1-cell C. elegans embryo. The latter finding demonstrates for the first time that although the anterior-posterior embryonic axis in C. elegans is specified by sperm, the sperm pronucleus itself is not required. Also unaffected is the completion of oocyte meiosis, formation of an impermeable eggshell, migration of the oocyte pronucleus, and the separation and expansion of the sperm-contributed centrosomes. Our investigation of these mutants confirms that, in C. elegans, neither the sperm chromatin mass nor a sperm pronucleus is required for spermiogenesis, proper egg activation, or the induction of anterior-posterior polarity.

Mutations in the alpha-tubulin 67C gene specifically impair achiasmate segregation in Drosophila melanogaster.

Drosophila melanogaster oocytes heterozygous for mutations in the alpha-tubulin 67C gene (alphatub67C) display defects in centromere positioning during prometaphase of meiosis I. The centromeres do not migrate to the poleward edges of the chromatin mass, and the chromatin fails to stretch during spindle lengthening. These results suggest that the poleward forces acting at the kinetochore are compromised in the alphatub67C mutants. Genetic studies demonstrate that these mutations also strongly and specifically decrease the fidelity of achiasmate chromosome segregation. Proper centromere orientation, chromatin elongation, and faithful segregation can all be restored by a decrease in the amount of the Nod chromokinesin. These results suggest that the accurate segregation of achiasmate chromosomes requires the proper balancing of forces acting on the chromosomes during prometaphase.

Hippocampal Neuronal Death following Deep Hypothermic Circulatory Arrest in Dogs: Involvement of Apoptosis

This study was undertaken to evaluate the histological nature of brain damage caused by deep hypothermic circulatory arrest during cardiopulmonary bypass. Total body cooling to 15°C and rewarming were performed with a conventional cardiopulmonary bypass technique using the femoral artery and vein. Dogs were assigned to one of three groups. In group 1 ( n = 4), cardiopulmonary bypass was maintained in a state of deep hypothermia (15°C) for 90 min, group 2 animals ( n = 5) underwent 60 min of deep hypothermic circulatory arrest at 15°C, and group 3 ( n = 6) underwent 90 min of deep hypothermic circulatory arrest at 15°C. All dogs were killed by perfusion fixation 72 h after cardiopulmonary bypass. The CA1 regions of the hippocampi were examined by light and electron microscopy. Biotinylated dUTP was used for nick-end labeling of apoptotic cells mediated by terminal deoxytransferase. No morphological change was observed in group 1 dogs, and very little in group 2 dogs. More severe neuronal damage was observed in group 3. The nuclei of many cells were shrunken and showed nick-end labeling. Dense chromatin masses were detected electron microscopically in the nuclei of CA1 pyramidal cells. Neuronal cell death observed in CA1 pyramidal cells 72 h after 90 min of deep hypothermic circulatory arrest at 15°C involves apoptosis. Therefore, according to this model, the maximum duration of deep hypothermic circulatory arrest should not be allowed to exceed 60 min.

Hippocampal neuronal death following deep hypothermic circulatory arrest in dogs: involvement of apoptosis

This study was undertaken to evaluate the histological nature of brain damage caused by deep hypothermic circulatory arrest during cardiopulmonary bypass. Total body cooling to 15°C and rewarming were performed with a conventional cardiopulmonary bypass technique using the femoral artery and vein. Dogs were assigned to one of three groups. In group 1 ( n=4), cardiopulmonary bypass was maintained in a state of deep hypothermia (15°C) for 90 min, group 2 animals ( n=5) underwent 60 min of deep hypothermic circulatory arrest at 15°C, and group 3 ( n=6) underwent 90 min of deep hypothermic circulatory arrest at 15°C. All dogs were killed by perfusion fixation 72 h after cardiopulmonary bypass. The CA1 regions of the hippocampi were examined by light and electron microscopy. Biotinylated dUTP was used for nick-end labeling of apoptotic cells mediated by terminal deoxytransferase. No morphological change was observed in group 1 dogs, and very little in group 2 dogs. More severe neuronal damage was observed in group 3. The nuclei of many cells were shrunken and showed nick-end labeling. Dense chromatin masses were detected electron microscopically in the nuclei of CA1 pyramidal cells. Neuronal cell death observed in CA1 pyramidal cells 72 h after 90 min of deep hypothermic circulatory arrest at 15°C involves apoptosis. Therefore, according to this model, the maximum duration of deep hypothermic circulatory arrest should not be allowed to exceed 60 min.

Apoptosis in the early involuting stellate reticulum of rat molar tooth germs

When the enamel organ of the rat tooth germ is fully developed at the tip of the prospective cusp, amelogenesis begins, and at this site the overlaying stellate reticulum begins its involution. During the involution process, there is a gradual decrease in intercellular spaces, invasion by blood vessels, appearance of macrophage-like cells and reduction in the number of stellate reticulum cells. Since reduction or disappearance of cells during embryonic development in organs and tissues has been shown to occur by apoptosis, we decided to examine early involuting regions of the stellate reticulum in the hope of detecting apoptosis. For this purpose, upper first molars of Wistar newborn rats aged 1 and 3 days were fixed in formaldehyde for the TUNEL method and in glutaraldehyde-formaldehyde for light and electron microscopy. Paraffin sections revealed TUNEL-positive structures, i.e. brown-yellow-stained bodies, in the central portion of the stellate reticulum, and next to the outer enamel epithelium and stratum intermedium. Examination of ultrathin sections confirmed the TUNEL findings: some stellate reticulum cells showed nuclei containing crescent-like electron-opaque condensed masses of peripheral chromatin, typical of apoptosis. Also, apoptotic bodies of various sizes and appearances were frequently observed within stellate reticulum cells. We should like to suggest that apoptosis is associated with the reduction in the number of cells during regression of the reticulum.

Nuclei of symbiotic arbuscular mycorrhizal fungi as revealed by in vivo two-photon microscopy.

The present work reports the results obtained from in vivo studies on the distribution and behavior of nuclei of two arbuscular mycorrhizal (AM) fungi growing in symbiosis with tomato root organ cultures (AM monoxenic cultures). Upon staining with 4',6-diamidino-2-phenylindole and two-photon microscopy (2PM) observations, symbiotic thick runner hyphae appeared mostly opaque to 2PM and did not reveal nuclei within them; thin runner hyphae showed dimly stained nuclei along them, whereas nuclei were clearly visible within the branches of the so-called branched absorbing structures. When visible, nuclei appeared anchored laterally at regular intervals along the symbiotic AM extraradical hyphae. Other nuclei migrate through the hyphal central core; this migration occurs in pulses. Simultaneous observations on different areas of extraradical AM mycelium revealed the existence of lysed compartments along the fungal hyphae, containing nuclei remnants and/or chromatin masses. All these results give new insights in (i) the differential permeability of AM hyphae in the symbiotic versus the asymbiotic state; (ii) the behavior and distribution of nuclei along the symbiotic extraradical mycelium; (iii) the occurrence of ageing events within the AM fungal colony; and (iv) the existence of "healing" mechanisms aiming to restrict the damage induced by such ageing or lytic events. An AM fungal strategy for hyphal survival under adverse conditions is also suggested.