Cytochemical and Radioautographic Identification of Cells Induced to Synthesize Hemoglobin

In order to facilitate the identification of bone marrow cells in which hemoglobin synthesis is initiated, erythropoiesis was first suppressed in guinea pigs through the induction of posthypoxic polycythemia, and then it was restimulated by bleeding and reexposure to hypoxia. Hemoglobin synthesis was detected with ⁵⁵Fe incorporation on radioautographs, and its presence was demonstrated in the light microscope with the benzidine reaction and absorption of monochromatic light at λ 4046 Å. In the electron microscope, hemoglobin was detected in the cytoplasm by a general increase in electron density after treating the tissue with diaminobenzidine (DAB) and OsO₄. Densitometric measurements were carried out on electronmicroscopic negatives, using reticular cell cytoplasm as a base line. In normal marrow, proerythroblasts were the earliest cells in which hemoglobin could be detected, but during the early phase of erythropoietic stimulation, hemoglobin was demonstrated in transitional cells with all the methods employed. Without the specific demonstration of hemoglobin, these cells could not be recognized morphologically as erythroblasts nor could they be distinguished from the precursors of bone marrow small lymphocytes. Transitional cells were numerous in the marrow at the time of stimulation, and 40 hr later a small number of them were labeled with ⁵⁵Fe and synthesized hemoglobin in detectable amounts. Proerythroblasts were absent at the time of the stimulus, and when they reappeared the majority were benzidine or DAB positive and had incorporated ⁵⁵Fe. The findings suggest that progenitor cells of erythroblasts are among the basophilic members of the transitional cell population, and erythropoietic stimulation induces hemoglobin synthesis in them. The relationship of these cells to the progenitors of other hemopoietic cells, as well as to the pluripotent stem cell, is discussed.