Comparative ultrastructure of maturing toad (Bufo ictericus) and rabbit (Oryctolagus cuniculus) erythroid cells with regard to hemoglobin biosynthesis

1. Toad and rabbit maturing erythroid cells were comparatively analysed with regard to their ultrastructural modifications involved in hemoglobin (Hb) biosynthesis. 2. The mitochondrial inner membrane differentiates to a lamellated body that, successively, gives rise to prehemosomal vesicles, prohemosomes, and to hemoglobinized organelles called hemosomes. 3. The prehemosomal vesicle involves ferruginous inclusions, taken as iron sources for heme biosynthesis, as well as the polypeptide globin chains, assembling themselves in the course of volume reduction. 4. From the prohemosomal stage onwards, where possibly heme biosynthesis occurs, hemosomes are formed; these organelles are presumably sites where the final Hb biosynthesis could take place. 5. All development stages leading to hemosome formation are similar in toad and rabbit erythroid cells, except that, in the toad, the structural prohemosome characteristics persist in hemosomes. 6. Through toad erythroid cell fractionation and electrophoresis of the organelle lysate supernatant, a wide and a weak cytoplasmic Hb bands were obtained; the latter coincides with the intraorganellar Hb band.     

Mitochondrial kinetics during amphibian erythropoiesis related to haeme synthesis

Flow cytometry, light and epifluorescence microscopies and transmission electron microscopy were used to follow the mitochondrial kinetics during amphibian erythropoiesis. A similar behaviour in response to the induction of anaemia was observed in the diploid Bufo ictericus and the tetraploid Odontophrynus americanus. A high cellular activity was observed ten days after haemolytic anaemia induced by phenylhydrazine, based on the higher Rhodamine 123 uptake by the erythroid cells. In addition, the more intense expression of the mitochondrial enzyme cytochrome oxidase, isocitrate and succinic dehydrogenases were cytochemically detected at this stage. This suggests that erythroid cell mitochondria, at this time, could be in a more active functional state than at other stages. In both species, mitochondrial plasticity was observed during cell maturation. A progressive loss of oxidation-reduction enzyme expression seemed to follow changes at the mitochondrial cristae morphology, from transverse to longitudinal form, mainly at the 20th day of recovery from anaemia, possibly related to a natural loss of function. The presence of these mitochondrial enzymes in mitochondrion-like organelles also favours their participation in the haeme synthesis, although with a reduced expression, since this suggests the presence of a complete and active enzymatic complex in these modified organelles. This also supports the idea that all these organelles are mitochondria in distinct metabolic stages, and not mitochondrion-like organelles or haemosomes, as proposed by some authors.     

Hemoglobin biosynthesis enhancement following mitochondrial membrane growth and differentiation

1. A quantitative increase of organelles in early reticulocytes has been observed compared to that found in late erythroblasts of the peripheral rabbit embryo blood. 2. The increase is due to the formation of hemosomes, organelles taken as sites for final hemoglobin (Hb) biosynthesis or where the assembly of heme and globin polypeptides could occur. 3. These organelles derive indirectly from mitochondria whose internal membrane grows concomitantly to its differentiation, originating lamellated bodies that modify successively to prehemosomal vesicles, prohemosomes and hemosomes. 4. The occurrence of membrane synthesis for the formation of lamellated bodies could explain the increase of organelles per cell and, thereby, the enhancement of the Hb biosynthesis rate in peripheral embryo blood in relation to this biosynthesis rate in the liver, as had been biochemically ascertained by other authors.     

Co-clustering of Golgi complex and other cytoplasmic organelles to crescentic region of half-moon nuclei during apoptosis

Early apoptosis is defined by stereotypic morphological changes, especially evident in the nucleus, where chromatin condenses and compacts, and assumes a globular, half-moon or crescent-shaped morphology. Accumulating evidence suggests that cytoplasmic organelles such as mitochondria and the Golgi complex are major sites of integration of pro-apoptotic signaling. In this study, cytoplasmic organelles including Golgi complex, mitochondria, endosomes, lysosomes, and peroxisomes were shown to condense at the same unique region adjacent to the crescentic nucleus during a relatively early stage of apoptosis induced by staurosporine or other agents. The co-clustering phenomenon may be caused by shrinkage of cytoplasm during apoptosis although cytoskeletal markers actin and tubulin were not condensed and appeared excluded. These data suggest the co-clustering of cytoplasmic organelles plays an interesting role during the progression of the apoptotic process. It is possible that modification of pro-apoptotic proteins may arise as a result of the interplay of these cytoplasmic organelles.     

Autophagy in embryonic erythroid cells: its role in maturation

Yolk sac-derived embryonic erythroid cells differentiate synchronously in the peripheral blood of Syrian hamster. The stage of differentiation on day 10 of gestation is equivalent to polychromatophilic erythroblast stage and that on day 13 is equivalent to the reticulocyte stage in adult animals. The cytoplasm of embryonic erythroid cells became scant and devoid of most organelles on day 12 of gestation. In addition, there were very few non-erythroid cells in circulation before day 13. Thus the embryonic erythroid cells serve a pure and synchronous system to study the mechanisms of terminal differentiation. The number of mitochondria in the embryonic erythroid cells decreased to about 10% of the initial number during the period between day 10 and day 12 of gestation. In contrast, the frequency of autophagy of mitochondria increased 4.6-fold in the same period. The cytochrome c content of the cell decreased as the mitochondria became extinct. However, release of cytochrome c into the cytoplasm was not detectable through day 10-13 of gestation, suggesting that the mitochondria were digested within a closed compartment. Decomposed mitochondria and ferritin particles were detected in lysosomes by electron microscopy on and after day 12 of gestation, which also suggested digestion in a closed compartment. Mitochondrial ATP synthase subunit c, which is known to be a protease-refractory protein, was retained in the cells even after the disappearance of mitochondria, indicating that most of the mitochondria were not extruded from the cells. The digestion of mitochondria in autolysosomes may allow the cells to escape from rapid apoptotic cell death through concomitant removal of mitochondrial death-promoting factors such as cytochrome c.     

Mitochondria,hemosomes and hemoglobin biosynthesis

Erythroid cells of the liver and peripheral blood of rabbit embryos, as welt of bone-marrow and peripheral blood of adult rabbits with phenylhydrazine-induced hemolytic anemia, were analysed ultrastructurally to investigate the formation of hemosomes, organelles suggested to be sites of heme integration into the four globin polypeptides. After the incorporation of iron-containing material, free ferruginous inclusions appear. Mitochondria apparently give rise to lamellated bodies whose double lamellae expand for the captation of the ferruginous inclusions, a source of iron for heme synthesis, and globin polypeptidic chains already synthesized in the diffusely distributed polysomes. The expanding lamellae return, so that prehemosomal vesicles containing ferruginous material and globin are formed. Through invaginations of the inner membrane and a possible rotational movement of these vesicles the beginning of prohemosome formation takes place concomitant with the occurrence of heme synthesis. A structural rearrangement of prohemosomes occurs, and typical hemosomes containing hemoglobin molecules develop, whose content spreads throughout the cytoplasm by disruption of the organelle membranes.     

Mitochondrial clearance by autophagy in developing erythrocytes: Clearly important,but just how much so?

Erythrocytes are anucleated cells devoid of organelles. Expulsion of the nucleus from erythroblasts leads to the formation of reticulocytes, which still contain organelles. The mechanisms responsible for the final removal of organelles from developing erythroid cells are still being elucidated. Mitochondria are the most abundant organelles to be cleared for the completion of erythropoiesis. Macroautophagy, referred to as autophagy, is a regulated catabolic pathway consisting of the engulfment of cytoplasmic cargo by a double membraned-vesicle, the autophagosome, which typically then fuses to lysosomal compartments for the degradation of the sequestered material. Early electron microscopic observations of reticulocytes suggested the autophagic engulfment of mitochondria (mitophagy) as a possible mechanism for mitochondrial clearance in these. Recently, a number of studies have backed this hypothesis with molecular evidence. Indeed, the absence of Nix, which targets mitochondria to autophagosomes, or the deficiency of proteins in the autophagic pathway lead to impaired mitochondrial clearance from developing erythroid cells. Importantly, however, the extent to which the absence of mitophagy affects erythroid development differs depending on the model and gene investigated. This review will therefore focus on comparing the different studies of mitophagy in erythroid development and highlight some of the remaining controversial points.     

On a possible relationship between bacterial endospore formation and the origin of eukaryotic cells

The acquisition of intracellular organelles, including mitochondria and plastids and a membrane-bounded nucleus, have been postulated to be key events in the development of the eukaryotic from the prokaryotic ancestral cell. The two major hypotheses to account for such acquisitions are: (1) primitive cells originally obtained organelles by engulfing free-living prokaryotes which then entered into symbiotic association (“endosymbiosis”) with them; (2) organelles arose through the engulfment by the primitive cell of part of its own cytoplasm. To some extent, the former hypothesis has received most support, because endosymbiosis is known to occur in extant organisms, whilst the latter hypothesis has received less support, because cytoplasmic engulfment by prokaryotes is not now thought to occur. However, during the process of endospore formation by extant bacteria, the protoplast within the single cell is observed to divide in a unique manner such that the cell in effect engulfs a portion of its own cytoplasm. The process is strikingly similar to the engulfment suggested by the second hypothesis to have initiated the evolution of eukaryotes. The engulfed cytoplasm is bounded by a double membrane within the “mother cell” and contains enzymes, ribosomes and a complete genome. In many respects this parallels the supposed primitive eukaryotic state and, it is argued, confers potential advantages on the cell, particularly through the control that the “mother cell” can exert on the enclosed compartment. It is hypothesized that bacterial endospore formation is therefore one product of evolution from an early engulfment event that led also to the development of complex eukaryotic cells.     

Ultrastructure of maturing fish (Oreochromis niloticus) and snake (Waglerophis merremii) erythroid cells with regard to hemoglobin biosynthesis

• 1.1. Fish and snake immature erythrocytes were submitted to a comparative ultrastructural study, analysing changes in organelles involved in hemoglobin (Hb) biosynthesis.
• 2.2. Iron uptake occurs probably via transferrin, and ferruginous compounds accumulate as siderosomes, taken as iron sources for heme biosynthesis, later on caught by a double lamella.
• 3.3. Mitochondrial membrane of the inner camera differentiates to lamellated bodies that, sucessively, give rise to expansions for ferruginous material and globin chains captation, constituting prehemosomal vesicles, which become condensed vesicles, followed by prohemosomes.
• 4.4. Through an internal membrane rearrangement, prohemosomes change to hemosomes wherein, hypothetically, heme and the globin chains assembly may occur.
• 5.5. In both fish and snake erythroid cells, all stages for hemosomegenesis are similar to the stages found in erythroid cells of other vertebrate species, including humans, except that fish cells often present single organelles of still unknown function, void of internal membrane.
• 6.6. Through electrophoresis of the respective supernatants obtained after osmotical lysis of the organellar fractions, it was shown that fish hemosomes contain three Hb patterns, while snake hemosomes present two patterns.

     

Oxidation and denaturation of hemoglobin encapsulated in liposomes

A study was made of the in vitro stability of hemoglobin-containing liposomes (‘hemosomes’) prepared from phosphatidylcholines, equimolar cholesterol and red cell lysate by the hand-shaking and ether-injection methods. Absorption spectra indicated hemichrome formation in ‘hemosomes’ prepared by the ether-injection technique, and increased oxidation of hemoglobin in hand-shaken ‘hemosomes’. The denaturation of hemoglobin in ether-injection ‘hemosomes’ was increased if the initial methemoglobin content of the hemolysate, or the temperature of preparation was elevated. It was slower if liposomes were prepared under either N₂ or CO, or if the radical scavenger 1,3-diphenylisobenzofuran was added with the ether. Egg phosphatidylcholine and synthetic saturated phospholipids gave the same results. With hand-shaken ‘hemosomes’ the oxidized product was primarily methemoglobin, and oxidation could be inhibited by using saturated phosphatidylcholines instead of egg phosphatidylcholine. Lysophosphatidylcholine levels were higher and arachidonic acid levels lower in egg phosphatidylcholine ‘hemosomes’ than in equivalent liposomes containing no hemolysate. The ‘hemosome’ seems to be a suitable model for the study of hemoglobin-lipid membrane interactions and the resulting hemoglobin denaturation process.     

CYTOLOGICAL BASIS FOR CYTOPLASMIC INHERITANCE IN PSEUDOTSUGA MENZIESII. II. FERTILIZATION AND PROEMBRYO DEVELOPMENT

Douglas fir (Pseudotsuga menziesii [Mirb.] Franco) ovules were used to study male gamete formation, insemination of the egg, and free nuclear and cellular proembryo development. Two male nuclei form as the pollen tube either reaches the megaspore wall or as it enters the archegonial chamber. No cell wall separates them. They are contained within the body-cell cytoplasm. A narrow extension of the pollen tube separates the neck cells and penetrates the ventral canal cell. The pollen tube then releases its contents into the egg cytoplasm. The two male gametes and a cluster of paternal organelles (plastids and mitochondria) migrate within the remains of the body-cell cytoplasm toward the egg nucleus. Microtubules are associated with this complex. The leading male gamete fuses with the egg nucleus. The zygote nucleus undergoes free nuclear division, but the cluster of paternal organelles remains discrete. Free nuclei, paternal and maternal nucleoplasm, maternal perinuclear cytoplasm, and the cluster of paternal organelles migrate en masse to the chalazal end of the archegonium. There, paternal and maternal organelles intermingle to form the neocytoplasm, the nuclei divide, and a 12-cell proembryo is formed. The importance of male nuclei or cells, the perinuclear zone, and large inclusions in cytoplasmic inheritance are discussed in the Pinaceae and in other conifer families. This completes a two-part study to determine the fate of paternal and maternal plastids and mitochondria during gamete formation, fertilization, and proembryo development in Douglas fir.     

Fine structure of developing hagfish erythrocytes with particular reference to the cytoplasmic organelles

Cytological changes accompanying the maturation of erythrocytes in the “Pacific hagfish” (Eptatretus stoutii) were studied. Great numbers of immature and mitotically dividing red blood cells in the peripheral circulation of the hagfish appear to indicate that extensive differentiation and proliferation occurs in the blood stream of this animal. The immature erythrocytes contained mitochondria, Golgi membranes, centrioles, microtubules and a high density of ribosomes in the cytoplasm. Intermediate stages revealed lysosomes in the cytoplasm. With progressive differentiation the hagfish erythrocytes accumulate hemoglobin and lose most of their cytoplasmic organelles. The various cytoplasmic organelles are apparently lost through a degradation process brought about by lysosomal autolysis. The undigested products of degradation such as mitochondrial and other intercellular membranes are apparently extruded by way of the plasma membrane. The plasma membrane of young as well as mature erythrocytes display evidence of intense pinocytotic activity. The nucleolus undergoes a reduction in size with progressive maturation. The cytoplasm of mature erythrocytes consists predominantly of hemoglobin. An equatorial microtubular marginal band is identifiable in differentiating erythrocytes.     

CYTOPLASMIC LABEL FOLLOWING TRITIATED THYMIDINE TREATMENT OF ALLIUM CEPA L. ROOTS : Cytochemical and Electron Microscope Study

Tritiated thymidine routinely labels onion root cytoplasm during most of the cell cycle. One-third of this label could be cytochemically identified as DNA. The balance of the label was not RNA or a lipid, or attributable to labeled impurities in thymidine-³H. In electron microscope radioautographs one-third of the cytoplasmic silver grains was over organelles, presumably mitochondria and plastids. The other two-thirds of the silver grains in electron micrographs was distributed widely, 41% over ground cytoplasm and 10% over cell walls-cell membranes. Snake venom phosphodiesterase (SVDase) extracted a cytoplasmic fraction not degraded by DNase, and did not appear to extract nuclear DNA. The SVDase-extractable fraction may be DNA or a thymidine 5'-phosphoryl group in an ester linkage with another hydroxylic compound. The nature of the nonextractable fraction is considered. Possibilities discussed are: (1) technical problems such as the binding of an acid-labile nuclear DNA in the cytoplasm; (2) non-DNA, such as breakdown products, and thymine compounds other than DNA; (3) DNA, not extractable because of the nature of its binding to other compounds or because it is a "core" resistant to DNase. Until the chemical nature of this nonextractable fraction is known, cytoplasmic label following thymidine-³H treatment cannot necessarily be considered DNA, nor the assumption made that thymidine-³H exclusively labels DNA.     

Ultrastructural changes in the spermatogonia and spermatocytes of Poecilia reticulata during spermatogenesis

Summary The structure of guppy (Poecilia reticulata) spermatogonia and spermatocytes has been studied using electron microscopy. The spermatogonia, situated at the apex of the seminiferous tubule, are almost all surrounded by a network of Sertoli cells; they have very diffuse chromatin and one or two large nucleoli. The cytoplasm contains relatively few organelles, although annulate lamellae are found. The mitochondria have few cristae and are concentrated at one pole of the cell; they are sometimes found with intermitochondrial cement. These spermatogonia are separated from each other, having no intercellular bridges or inclusion in Sertoli cells, and are relatively undifferentiated; they correspond to stem cells. The spermatogonia beneath the apex are organized into cysts. First-generation spermatogonia are more dense and heterogeneous, their nuclei becoming smaller and their chromatin becoming denser during successive generations. In spermatocytes, the synaptinemal complex exists as a modified form until metaphase. The concentration of organelles in the cytoplasm increases and the organelles become more diversified as spermatogenesis progresses. Many cytoplasmic bridges are observed (several per cell), indicating that the cells remain in contact after several divisions. These changes in germ cell structure have been related to some of the characteristic features of spermatogenesis in guppy, e.g. the large number of spermatogonial generations and the complexity of spermiogenesis.     

Organic anion transport in macrophage membrane vesicles

Cells of the J774 mouse macrophage-like cell line possess organic anion transporter that transport fluorescent dyes such as Lucifer Yellow out of the cytoplasmic matrix of the cells; the dye is both sequestered in endosomes and secreted into the extracellular medium. Lucifer Yellow that is sequestered within endosomes is subsequently delivered to the lysosomal compartment. In the present studies we demonstrated that probenecid inhibited removal of Lucifer Yellow from the soluble cytoplasm and sequestration into membrane bound organelles by quantitating Lucifer Yellow fluorescence in both soluble and membrane-associated fractions of J774 cells. In addition, we examined the uptake of Lucifer Yellow into isolated subcellular organelles derived from J774 cells. Lucifer Yellow transport in the organellar fraction of J774 cell homogenates was temperature- and pH-dependent and did not require ATP. Subcellular organelles from J774 cells were fractionated into endosome- and lysosome-enriched fractions by Percoll density gradient centrifugation. Lucifer Yellow was preferentially taken up by vesicles of the endosome-enriched fraction, and this transport was inhibited by probenecid. These studies provide direct evidence that probenecid inhibits Lucifer Yellow transport out of the cytoplasmic matrix and into cytoplasmic vacuoles in J774 cells and that organic anion transport in isolated organelles derived from J774 cells occurs preferentially in endosome, rather than in lysosome-enriched fractions; they suggest that Lucifer Yellow is carried across membranes via a secondary active transport process that requires proton symptom or hydroxyl anion antiport.     

LIGHT AND ELECTRON MICROSCOPIC STUDIES OF THE FUSION CELL IN ASTEROCOLAX GARDNERI SETCH. (RHODOPHYTA,CERAMIALES)12

The fusion cell in Asterocolax gardneri Setch, is a large, multinucleate, irregularly-shaped cell resulting from cytoplasmic fusions of haploid and diploid cells. Subsequent enlargement takes place by incorporating adjacent gonimoblast cells. The resultant cell consists of two parts—a central portion of isolated cytoplasm, surrounded by an electron dense cytoplasmic barrier, and the main component of the fusion cell cytoplasm surrounding the isolated cytoplasm. The fusion cell contains many nuclei, large quantities of floridean starch, endoplasmic reticulum, and vesicles, but few mitochondria, plastids and dictyosomes. The endoplasmic reticulum forms vesicles that apparently secrete large quantities of extracellular mucilage which surrounds the entire carposporophyte. The isolated cytoplasm also is multinucleate but lacks starch and a plasma membrane. Few plastids, ribosomes and mitochondria are found in this cytoplasm. However, numerous endoplasmic reticulum cisternae occur near the cytoplasmic barrier and they appear to secrete material for the barrier. In mature carposporophytes, all organelles in the isolated cytoplasm have degenerated.     

Nonuniform distribution of sodium in the rat hepatocyte

The volume of the nucleus, endoplasmic reticulum (including Golgi complex), mitochondria, and cytoplasmic ground substance was measured in rat hepatocytes by stereological methods. The Na content was also measured by flame photometry. Variations in Na content correlated significantly with variations in volume of nucleus and endoplasmic reticulum. From the correlation parameters, Na concentrations were estimated as follows: nucleus, 108 mM; endoplasmic reticulum (ER) (including Golgie complex) 27 mM; cytoplasm (including and remaining organelles) 16 mM.     

Microgametogenesis in Plumbago zeylanica (Plumbaginaceae). 2. Quantitative cell and organelle dynamics of the male reproductive cell lineage

Quantitative cell and organelle dynamics of the male gamete-producing lineage of Plumbago zeylanica were examined using serial transmission electron microscopic reconstruction at five stages of development from generative cell inception to sperm cell maturity. The founder population of generative cell organelles includes an average of 3.88 plastids, 54.9 mitochondria, and 3.7 vacuoles. During development the volume of the pollen grain increases from 6,200 μm³ in early microspores to 115,000 μm³ at anthesis, cell volume of the male germ lineage decreases more than 67% from 362.3 μm³ to 118.4 μm³. By the time the generative cell separates from the intine, plastid numbers increase by >600%, mitochondria by 250%, and vesicles by 43 times. A cellular projection elongates toward and establishes an association with the vegetative nucleus; this leading edge contains plastids and numerous mitochondria. When the generative cell completes its separation from the intine, organellar polarity is reversed and plastids migrate to the opposite pole of the cell. Cytoplasmic microtubules are common in association with cellular organelles. Plastids accumulate at the distal end of the cell as a linked mass, apparently adhered by lateral electron dense regions. Before division of the highly polarized generative cell, plastids decrease in number by 16%, whereas mitochondria increase by ∼90% and vacuoles increase by ∼140% from the prior stage. After mitosis, the resultant sperm cells differ in size and organelle content. The sperm cell associated with the vegetative nucleus (S_vn) contains 62.7% of the cytoplasm volume, 87% of the mitochondria, 280.4 vesicles (79% of those in the generative cell), and 0.6% of the plastids. At maturity, the S_vn mitochondria increase by 31% and the cell contains an average of 0.4 plastids, 158.9 vesicles, and 0.36 microbodies. The mature unassociated sperm (S_ua) contains 39.8 mitochondria (up 3.3%), 24.3 plastids (down 31%), 91.1 vesicles (up 54.9%), and 3.18 microbodies. The small number of organelles initially in the generative cell, followed by their rapid multiplication in a shrinking cytoplasm suggests a highly competitive cytoplasmic environment that would tend to eliminate residual organellar heterogeneity. Cell and cytoplasmic volumes vary as a consequence of fluctuations in the number and size of large vesicles or vacuoles, as well as loss of cytoplasmic volume by (1) formation of “false cells” involving amitotic cytokinesis, (2) “pinching off” of cytoplasm, and (3) dehydration of pollen contents prior to anthesis.     

A relative morphological evaluation of hemoglobin biosynthesis in peripheral blood reticulocytes of normal and anemic rabbits

1. Peripheral blood reiculocytes of normal and bled rabbits and of rabbits with phenylhydrazine-induced anemia, were morphologically analysed, through silver sections, for a relative evaluation of hemoglobin (Hb) biosynthesis activity. 2. Reticulocytes of maturation degrees within the range of 35-60 polysomes/microns2, were compared as to their mean numbers of hemosomes (sites of heme integration into the globin chains), and mitochondria (indirect precursors for hemosome formation). 3. The results on the mean numbers of hemosomes per reticulocyte section, correlated to several physiological data under those three conditions, suggested a close relationship between Hb biosynthesis activity and hemosome frequency. 4. In bled rabbits, reticulocytes showing a low mean number of hemosomes (means hB/section = 0.32), as compared to reticulocytes of normal rabbits (means hN/section = 0.70) and to reticulocytes of rabbits with hemolytic anemia (means hH/section = 2.10), gave rise to a new erythrocyte population characterized by a low Hb content. 5. Hb concentration differences were verified by confronting hematological data before bleeding with those obtained after the regression of anemia.