A cytochemical and radioautographic analysis of erythropoiesis at the ultrastructural level

Abstract

The morphogenetic events of erythroid differentiation in guinea pig bone marrow were studied by electron microscopy. The diaminobenzidine (DAB ) reaction and radioautographic detection of Fe55 incorporation permitted a more objective identification of erythroid cells than was possible in previous studies. With the combined use of these two methods morphological events of cell maturation could be correlated reliably with cytoplasmic hemoglobin content, thus providing a basis for the description of various maturation stages.The oxidation of DAB by hemoglobin coupled with OsOl treatment results in a generalized increase in cytoplasmic electron opacity proportional to the amount of hemoglobin present. Measurements of cytoplasmic density were carried out on electron microscopic negatives of DAB-reacted specimens that were not stained otherwise. Only those cells were regarded as erythroblasts in which the cytoplasm was at least twice as dense as in recognizable, differentiated, non-erythroid cells, using reticular cell cytoplasm in each specimen as the base line for the comparisons.In bone marrow cells, iron incorporation occurred overwhelmingly for hemoglobin synthesis. In normal marrow, blast cells which incorporated iron but did not contain hemoglobin in detectable amounts were defined as Stage I erythroid cells. They were morphologically of the same type as erythroblasts with the minimum amount of detectable hemoglobin (Stage 11). Cells in maturation stages of progressively increasing hemoglobin content were assigned to Stages I11 to V. Stage I11 erythroblasts correspond morphologically to proerythroblasts of light microscopic preparations. The DAB reaction in them was obvious without densitometric measurements but remained mottled.The morphological characteristics of each of the maturation stages are described with particular emphasis on the earliest erythroid cells, since the methods employed permit their identification with greater degrees of discrimination and objectivity than is possible with the light microscope or with conventional methods of electron microscopy.The DAB reaction was positive in the heterochromatin in Stages I11 and IV but it was negative in the pyknotic chromatin of erythroblasts in Stage V of maturation. The predominantly extended chromatin and also the large and complex nucleoli of earlier cells began to condense during Stage IV. Silver grains due to Fej5 disintegration were observed over the nuclei of erythroblasts in all maturational stages.The perinuclear cisterna was DAB-negative and it became wider as maturation progressed. It was bridged in places by DAB-negative material connecting the cytoplasm to euchromatic areas of the nucleus. DAB-positive substance was not seen in nuclear pores at any stage of maturation. At the time of extrusion, nuclei did not contain DAB-positive material but were occasionally labeled with Fe5j.

Hemopoietic cells in the liver and spleen of the embryonic and early postnatal mouse: A karyometrical observation

“…The liver at the first stage contained large cells with nuclear and nucleolar morphological features similar to yolk sac angioblasts (Sasaki and Matsumura, 1986a1, and, under light microscopy, the liver was seen to contain large hemopoietic cells with nuclear membrane invagination. According to the erythroid cell classification of Rosse and Trotter (1974), these hemopoietic cells could correspond to hemocytoblasts. Large hemopoietic cells with nuclei larger than 8 pm, including hemocytoblasts, may play a very important role in migration of embryo hemopoiesis from yolk sac to liver.…”

The liver hemopoietic environment: II. Peroxidase reactive mouse fetal liver hemopoietic cells

“…Type I11 cells had a more intensely reactive cytoplasm, were closely related morphologically to polychromatophilic erythroblasts, and possessed a positive nuclear component, possibly developing hemoglobin associated with the heterochromatin (Figs. 13, 14) (Tooze and Davies, 1963;Rosse and Trotter, 1974). Type IV cells were predominately nucleated normoblasts possessing a very dense cytoplasmic reaction and a nucleus devoid of reaction product (Figs.…”

“…This reaction has been termed a pseudoperoxidase reaction (Ackerman and Clark, 1971) which is attributable to the presence of heme proteins in the cytoplasm. The staging of peroxidase reactive erythropoietic cells and nonreactive hemopoietic cells was based on the criteria set forth for typing similar cells in the adult bone marrow, except that the intensity of cytoplasmic reaction was performed visually without the aid of a densitometer (Rosse and Trotter, 1974).…”

“…Recently, the application of cytochemical and immunocytochemical techniques utilizing enzymes a n d or enzyme-antibody complexes to identify cel-lular enzyme and antigen characteristics have proven useful in this effort (Dvorak et al, 1972;Trotter, 1974,1975;Westen and Bainton, 1979;Rouse et al, 1979). Ackerman (1968), Rosse and Trotter (1974), and Dvorak et al (1972) examined erythropoietic and myelopoietic microenvironments of adult bone marrow using endogenous peroxidase activity as a marker for cellular maturation in these lineages. It was determined that erythropoietic and myelopoietic foci were distinguishable and spatially segregated in the bone marrow (Rosse and Trotter, 1974;Weiss, 1976;Westen and Bainton, 1979).…”

“…Ackerman (1968), Rosse and Trotter (1974), and Dvorak et al (1972) examined erythropoietic and myelopoietic microenvironments of adult bone marrow using endogenous peroxidase activity as a marker for cellular maturation in these lineages. It was determined that erythropoietic and myelopoietic foci were distinguishable and spatially segregated in the bone marrow (Rosse and Trotter, 1974;Weiss, 1976;Westen and Bainton, 1979). Campbell (1980Campbell ( ,1982 found that hemopoietic cells formed specialized membrane junctions with stromal cell types in the bone marrow.…”