Spleen of Dasypus hybridus (Mammalia, dasypodidae): a light and electron microscopic study.

Armadillos are relictual mammals important as models for biomedical studies. They contain adaptative and primitive characteristics in both anatomical and physiological aspects. In this study we describe the splenic histology and cytology of the "mulita," Dasypus hybridus. Organ samples were processed for light and electron microscopy study. The microanatomy of the organ samples as well as their different cell types are described. The spleen is non-sinusoidal, with the typical arrangement for storage functions. White pulp is lightly diffuse. Red pulp is a meshwork of circulating, immunocompetent and hemopoietic cells. Differences with other studied members of the group are discussed. The general structure of the organ agrees with the semi-fossorial habit of the species. Persistence of myeloid activity in the adult suggests the existence of specific inductive functions of the stroma. Better knowledge of this fact may give further insight on the phylogeny of hemopoiesis.

Immunocytochemical mapping of the hemoglobin biosynthesis site in amphibian erythroid cells.

During the past 25 years, several studies have attempted to determine the site of integration of the heme and the four globin chains in vertebrate erythroid cells that is important in the formation of the hemoglobin molecule. Mitochondrion-like organelles or hemosomes were pointed out as responsible for this task. We performed several experiments to investigate this hypothesis. The intracellular distribution of hemoglobin in amphibian erythroid cells was detected by post-embedding immuno-electron microscopy, using a polyclonal anti-human hemoglobin-proteinA-gold complex. Hemoglobin mapping showed an intense labeling in the cell cytoplasm, but none in cytoplasmic structures such as endoplasmic reticulum, mitochondria, mitochondrion-like organelles, Golgi complex, ribosomes or ferruginous inclusions. The mitochondrial fraction obtained according to the protocol described for some authors, showed by ultrastructural examination that this fraction has a heterogeneous content, also composed by microvesicles rich in cytoplasmic hemoglobin, an artifact generated by mechanical action during cell fractionation. Thus, when this fraction is lysed and its content submitted to electrophoresis, hemoglobin bands would be found inevitably, causing false-positive results, erroneously attributed to hemoglobin content of mitochondrion-like organelles. Our data do not confirm the hypothesis that the final hemoglobin biosynthesis occurs inside mitochondrion-like organelles. They suggest that the hemoglobin molecule be assembled in the erythrocyte cytoplasm outside of mitochondria or hemosomes.

Hemosomegenesis and hemoglobin biosynthesis in vertebrates.

1. Ultrastructural observations on maturing rabbit embryo erythroid cells led to the finding of hemoglobinized organelles distinguishable from mitochondria due to their highly dense matrix, two or three longitudinally arranged double lamellae, and smaller diameters. Intraorganellar 50-60 A particles identical to those contained in the hemoglobinized cytoplasm were found. 2. Their hemoglobin (Hb) content was demonstrated by electrophoresis of the concentrated supernatant from the isolated, washed, and osmotically lysed organellar fraction. We have proposed that these organelles are the sites for heme integration into the globin (G) polypeptide chains and subunits assembly. The term hemosome has been suggested for such entities. 3. This hypothesis has been sustained by several analytical and experimental works based on the postulation that hemosomes should be found at higher frequencies where the Hb biosynthesis rate is more intensive, or where the induction of this biosynthesis is always dependent on the formation of hemosomes. 4. Maturing erythroid cells of the circulating embryo blood contain hemosomes in higher frequency than in liver erythroid cells, coinciding with the higher Hb biosynthesis rate in peripheral blood than in the liver. In bleeding anemia, the decay of Hb concentration parallels the reduction of the mean number of hemosomes per reticulocyte, in comparison with normal reticulocytes. 5. In HeLa cells and epithelial cultured cells induced to synthesize Hb, it was shown that this biosynthesis is ever concomitant with the formation of hemosomes and depends on the presence of erythropoietin, as occurs in erythroid cells. 6. Studies on hemosomegenesis and Hb biosynthesis experimentally effected in epithelial cultured cells, allowed the interpretation of the sequence of events leading to hemosome formation in maturing erythroid cells. Simultaneously with iron uptake, mitochondria differentiate to lamellated bodies and, successively, expansions rise for ferruginous compounds and G polypeptides gathering, followed by prehemosome vesicles formation, which condense and change to prohemosomes that afterwards evolve to hemosomes. 7. These dynamics, and organellar Hb have been detected in immature erythrocytes of mammalians, including humans, avians, reptilians, amphibians and representative fish specimens. It appears that these events occur in the erythrocytary maturation of all vertebrate classes.

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.

Ultrastructural aspects of the final stage of hemoglobin biosynthesis.

1. Electron microscopic observations on rabbit embryo, adult rabbit, guinea pig, and human immature erythroid cells showed characteristic hemoglobinized organelles distinguishable from mitochondria by their highly dense matrix, two or three longitudinally arranged double lamellae, and smaller diameters. 2. The presence of hemoglobin (Hb) within these organelles was also demonstrated by electrophoresis of the concentrated supernatant from the isolated, washed and osmotically lysed organelle fraction. The term hemosome has been suggested for these organelles because of their Hb content. We propose that they are the sites of heme integration into the four polypeptide globin chains. 3. The frequency of hemosomes is higher in the peripheral blood erythroid cells of embryos than in the liver erythroid cells, coinciding with the higher Hb synthesis rate in peripheral blood than in the liver. 4. Peripheral blood reticulocytes of rabbits with anemia induced by bleeding presented a lower hemosome frequency than normal reticulocytes. The decrease paralleled the decay of Hb biosynthesis activity. Moreover, Hb biosynthesis induced in HeLa cell and epithelial cell tissue cultures was always associated with the formation of hemosomes. 5. Hemosomegenesis was studied in epithelial tissue culture cells experimentally induced to synthesize Hb, allowing the identification of several stages of hemosome formation in erythroid cells. The morphological data suggest that mitochondria are successively modified to lamellated bodies, prehemosomal vesicles, prohemosomes and hemosomes. These organelles have also been detected in erythroid cells of representative specimens from other vertebrate classes.

Ultrastructural aspects of the final stage of hemoglobin biosynthesis

Electron microscopic observations on rabbit embryo, adult rabbit, guinea pig, and human immature erythroid cells showed characteristic hemoglobinized organelles distinguishable from mitochondria by their highly dense matrix, two or three longitudinally arranged double lamellae, and smaller diameters. The presence of hemoglobin (Hb) within these organelles was also demonstrated by electrophoresis of the concentrated supernatant from the isolated, washed and osmotically lysec organelle fraction. The term hemosome has been suggested for these organelles because of their Hb content. We propose that they are the sites of heme integration into the four polypeptide globin chains. The frequency of hemosomes is higher in the peripheral blood erythorid cells of embryos than in the liver erythroid cells, coinciding with the higher Hb synthesis rate in peripheral blood than in the liver. Peripheral blood reticulocytes of rabbits with anemia induced by bleeding presented a lower hemosome frequency than normal reticulocytes. The decrease paralleled the decay of Hb biosynthesis activity. Moreover, Hb biosynthesis induced in HeLa cell and epithelial cell tissue cultures was always associated with the formation of hemosomes. Hemosomegenesis was studied in epithelial tissue culture cells experimentally induced to synthesize Hb, allowing the identification of several stages of hemosome formation in erythroid cells. The morphological data suggest that mitochondria are successively modified to lamellated...

Dyserythropoiesis in iron deficiency and in beta-thalassemia.

Nuclear and cytoplasmic abnormalities were quantitated in bone marrow erythroblasts from 15 patients with iron deficient anemia, 5 beta-thalassemia homozygotes, 5 beta-thalassemia heterozygotes, 6 S/beta-thalassemia double heterozygotes and 9 controls. The frequency of dyserythropoietic changes in iron deficiency was 11.90 +/- 5.02% (mean +/- SD) which is significantly higher than 3.36 +/- 1.16% obtained for the control group. The degree of dyserythropoiesis was negatively correlated with hemoglobin level (rS = 0.757). The frequency of dyserythropoietic changes obtained for the beta-thalassemia heterozygotes (5.23 +/- 1.45%) and for S/beta-thalassemia (7.13 +/- 2.00%) was elevated compared with the controls (P less than 0.05 and P less than 0.01, respectively). The highest frequency of dyserythropoiesis (19.88 +/- 7.40%) occurred among beta-thalassemia homozygotes. In all cases studied the abnormalities were observed mainly in the late erythroblasts. In addition, a peculiar cytoplasmic inclusion was observed in Leishman-stained bone marrow or peripheral blood erythroblasts from beta-thalassemia homozygotes, which is probably the result of precipitation of excess alpha-chain. This abnormality of thalassemia erythroblasts in Leishman-stained smears had not been previously reported.

Congential dyserythropoietic anemia--type IV.

Congenital dyserythropoietic anemia is characterized by ineffective erythropoiesis and increased numbers of multinucleated red cell precursors in the marrow. This syndrome has been subclassified on the basis of morphologic differences in the red cell precursors. Type I is characterized by megaloblastoid erythropoiesis and macrocytosis; Type II, by normoblastic multinuclearity and normocytosis; and Type III, by frequent giant multinucleated erythroblasts and macrocytes. Type II is further distinguished from the other types by serologic and ultrastructural abnormalities. The patient presented in this report does not fit any of the above categories; her red cells are similar to Type II congenital dyserythropoietic anemia cells, but no characteristic ultrastructural or serologic abnormalities are present. It is suggested that this patient may represent an additional variant of congenital dyserythropoietic anemia, Type IV.